METHODS    OF 
ORGANIC    ANALYSIS 


METHODS    OF 


ORGANIC   ANALYSIS 


BY 


HENRY   C.    SHERMAN,    PH.D. 

\\ 
ADJUNCT    PROFESSOR    OF    ANALYTICAL    CHEMISTRY 

IN' 
COLUMBIA    UNIVERSITY 


Neto  ¥otfc 
THE    MACMILLAN    COMPANY 

LONDON  :    MACMILLAN  &  CO.,  LTD. 


All  rights  reserved 


Copyright  1905 
BY  HENRY   C.    SHERMAN 


PRESS  OF 

HE  NEW  ERA  PRINTING  COMPANY, 
LANCASTER,  PA. 


PREFACE. 

The  purpose  of  this  work  is  to  give  a  connected  introductory 
training  in  organic  analysis,  especially  as  applied  to  plant  and 
animal  substances  and  their  manufactured  products.  No  attempt 
is  made  to  touch  upon  all  important  branches  of  this  subject  but 
representative  topics  are  treated  in  considerable  detail  with  refer- 
ence both  to  analytical  methods  and  to  the  interpretation  of  results. 

The  greater  part  of  the  book  is  devoted  to  quantitative  methods 
for  food  materials  and  related  substances.  Standard  works  of  ref- 
erence and  the  publications  of  the  Association  of  Official  Agri- 
cultural Chemists  have  been  freely  used.  The  nomenclature 
adopted  in  these  publications  has  been  followed  as  closely  as  possi- 
ble. As  a  rule,  footnotes  show  the  original  sources  of  statements 
or  methods  included  in  the  text,  while  general  or  additional  refer- 
ences are  given  at  the  end  of  each  chapter.  The  references  have 
been  carefully  selected  and  are  believed  to  be  sufficient  to  put  the 
reader  in  touch  with  the  most  important  literature. 

The  descriptions  of  methods  were  written  primarily  for  the  use  of 
third-year  students  in  the  School  of  Chemistry,  Columbia  Univer- 
sity, and  therefore  presuppose  a  knowledge  of  inorganic  quantitative 
analysis,  elementary  organic  chemistry,  and  general  physics. 

The  writer  takes  pleasure  in  acknowledging  his  indebtedness  to 
Professor  Edmund  H.  Miller  for  helpful  advice  and  suggestions 
throughout  the  work,  and  to  Mr.  Roland  H.  Williams  for  assis- 
tance in  testing  methods  and  in  the  revision  of  parts  of  the  manu- 

script'  H.  C.  S. 

NEW  YORK,  July  i,  1905. 


141386 


TABLE  OF  CONTENTS. 

CHAPTER  I. 
INTRODUCTION. 

Ultimate  and  proximate  analysis I 

Preliminary  treatment  of  samples I 

Outline  of  ultimate  organic  analysis 2 

Preparation  and  analysis  of  ash '   4- 

CHAPTER    II. 

NITROGEN,  SULPHUR,  AND  PHOSPHORUS. 

Determination  of  nitrogen • 8 

Kj  eldahl  method 8 

Gunning- Arnold-Dyer  modification I o 

Method  for  nitrates  and  nitro-compounds 13 

Determination  of  sulphur 14 

Comparative  outline  of  methods 14 

Leibig's  alkali  method 16 

Osborne'  s  peroxide  method 1 8 

Berthelot's  oxygen  method  19 

Determination  of  phosphorus 20 

Alkali  methods 20 

Neumann's  acid  method 21 

CHAPTER  III. 

ALCOHOLS 23 

Ethyl  alcohol 24 

Detection  and  identification ;...  25 

Determination  by  the  specific  gravity  method 26 

Determination  by  the  boiling  point  method 32 

Determination  by  oxidation 33 

Detection  and  determination  of  homologous  alcohols 33 

Methods  of  stating  strength  of  alcohol  solutions 35 

Gly  cerol 36 

Determination  by  oxidation 37 

Determination  by  acetylation 39 

Determination  by  separation  and  weighing 40 

Examination  of  commercial  glycerol.. 42 

CHAPTER  IV. 

ALDEHYDES 46 

Formaldehyde 48 

Detection  and  identification 50 

vii 


viii  CONTENTS. 

Determination  by  oxidation 52 

Determination  by  condensation  reactions 54 

Determination  by  addition  reactions 55 

Additional  references 57 

CHAPTER    V. 

CARBOHYDRATES  —  GENERAL  METHODS. 

Occurrence  and  relations 58 

Solubilities 6 1 

Reactions  with  phenylhydrazine 63 

Preparation  and  properties  of  the  osazones 64 

Reduction  of  copper  solutions 67 

Fehling's  volumetric  method 69 

Defren's  gravimetric  method 72 

Kjeldahl's  gravimetric  method 74 

Barfoed'  s  cupric  acetate  method 75 

Reactions  with  acids 76 

Molisch'  s  tf-naphthol  reaction 76 

Furfurol  reaction  of  pentoses  and  pentosans 77 

Levulinic  acid  reaction  of  hexoses 78 

Oxidation  by  nitric  acid 79 

Hydrolysis  by  dilute  acids 80 

Rotation  of  polarized  light 81 

Measure  of  rotating  power — Specific  rotation 81 

Preparation  of  solutions  for  polarization 83 

Determination  of  angular  rotation ,  84 

Reference  books 85 

CHAPTER  VI. 

CARBOHYDRATES — SPECIAL  METHODS. 

Analysis  of  raw  sugar 86 

Polariscopic  examination 86 

Clerget's  method  for  sucrose 91 

Determination  of  reducing  sugars.. 92 

Determination  of  moisture  and  ash 93 

Official  methods  and  standards  of  purity 93 

Determination  of  sucrose  in  beets  and  cane 94 

Commercial  glucose 96 

Official  definitions  and  standards  of  purity 96 

Analysis  by  Wiley's  method 97 

Analysis  by  other  methods 98 

CHAPTER  VII. 

CARBOHYDRATES  —  SPECIAL  METHODS  (Continued}. 

Determination  of  starch...  100 


CONTENTS.  ix 

Method  of  direct  acid  hydrolysis i  oo 

Method  of  digestion  with  diastase  or  saliva 103 

Comparison  of  results 105 

Determination  of  starch  in  meat  products 105 

Additional  references 1 06 

Separation  of  carbohydrates  in  cereal  products 107 

Determination  of  reducing  sugars,    sucrose,   dextrin,  starch, 

pentosans,  and  cellulose 107 

Determination  of  maltose,  dextrin,  and  starch  in  malted  cereal  108 

References  to  other  special  methods 109 

Substances  rich  in  sucrose  or  invert  sugar 109 

Artificial  mixtures  containing  lactose 110 

Animal  tissues  and  fluids  other  than  milk 1 1 1 

CHAPTER  VIII. 

ACIDS 112 

Acetic  acid  and  acetates 112 

Determination  of  acetic  acid  in  calcium  acetate 112 

Separation  of  acetic  acid  from  its  homologues 114 

Vinegar 115 

Determination  of  constituents 1 1 6 

Determination  of  source 117 

Official  standards 118 

References 1 1 8 

Fatty  acids 119 

Acids  of  the  stearic  series 119 

Acids  of  the  oleic  series 120 

Acids  of  the  linoleic  series 122 

Acids  of  the  linolenic  series 122 

Hydroxy-acids 122 

Separation  of  fatty  acids 123 

CHAPTER  IX. 

OILS,  FATS,  AND  WAXES  —  GENERAL  METHODS. 

Properties  of  fats  and  fatty  oils 125 

Analytical  methods 126 

Saponification  or  Koettstorfer  number 127 

Hehner  number 1 30 

Reichert-Meissl  number 1 30 

Iodine  or  Hiibl  number 130 

Maumene  number  —  Specific  temperature  reaction 1 36 

Acetyl  number 138 

Specific  gravity 140 

Index  of  refraction 141 

Melting  and  solidifying  points  —  Titer  test 142 


x  CONTENTS. 

Viscosity 143 

Heat  of  combustion 143 

Alcohols  of  the  fats  and  waxes  (unsaponifiable  matter). 144 

Alcohols  of  the  saturated  series 144 

Cholesterol  and  related  alcohols 145 

Reference  books 145 

Table  of  analytical  properties  of  oils,  fats,  and  waxes 146 

CHAPTER  X. 

FATTY  OILS —  SPECIAL  METHODS. 

Salad  oils 148 

Analytical  properties  of  olive  oil 149 

Detection  of  cottonseed  oil 150 

Detection  of  arachis  (peanut)  oil 152 

Detection  of  sesame  oil 153 

Detection  of  rapeseed  oil 154 

Detection  of  maize,  poppy-seed,  and  lard  oils 155 

Drying  oils 157 

Analytical  properties  of  linseed  oil 157 

Adulterants  and  methods  of  detection 158 

Hexabromide  test 1 60 

Oils  altered  by  age  or  oxidation 1 6 1 

Examination  of  an  unknown  oil 162 

Additional  references 1 63 

CHAPTER  XI. 

BUTTER 1 66 

Determination  of  water,  fat,  curd,  and  ash 166 

Examination  of  butter  fat 168 

Preparation 168 

Reichert-Meissl  or  Reichert-Wollny  number 1 68 

Specific  gravity  and  saponification  number 171 

Insoluble  fatty  acids  —  Hehner  number 171 

Iodine  number 172 

Melting  point  —  Wiley's  method 172 

Additional  determinations 173 

Composition  of  butter  fat -. .  1 74 

Variations  and  relations  of  analytical  properties 175 

Detection  of  oleomargarine 176 

Detection  of  cocoanut  fat 177 

Additional  references 178 

CHAPTER    XII. 
SOAPS  AND  LUBRICANTS. 

Analysis  of  commercial  soaps 180 


CONTENTS.       .  xi 

Determination  of  constituents 1 80 

Calculation  and  interpretation  of  results  187 

References 188 

Examination  of  lubricating  greases 189 

Examination  of  lubricating  oils  1 89 

Determination  of  constituents 1 90 

Viscosity  < 192 

Acidity 1 94 

Cold  test  and  chilling  point 195 

Flashing  and  burning  points 196 

Additional  determinations  197 

References 198 

CHAPTER    XIII. 
PROTEIDS  AND  CEREALS. 

Proteids  and  related  compounds 1 99  / 

Simple  proteids 1 99 

Combined  proteids   201 

Albuminoids  (albumoids,  proteoids,  gelatinoids) 202 

Other  nitrogen  compounds  —  Terminology  202 

General  reactions  of  proteids 203 

Separation  of  proteids  from  simpler  nitrogen  compounds  and 

from  each  other 206 

Cereals  and  other  grains  —  Mill  products 208 

Determination  of  moisture  and  fat 208 

Determination  of  nitrogen  compounds  210 

Determination  of  crude  fiber  and  of  ash  211 

Additional  tests  and  determinations  212 

Interpretation  of  results    212 

Reference  books   214 

CHAPTER   XIV. 
MILK. 

Normal  composition 215. 

Taking  and  preservation  of  samples 217 

Preliminary  or  partial  examination  218 

Determination  of  specific  gravity 218 

Volumetric  determination  of  fat 219 

Calculation  of  solids  from  specific  gravity  and  fat. 220 

Determination  of  fat,  proteids,  milk  sugar,  and  ash 220 

Total  solids  and  ash  220 

Fat  — Gravimetric  determination  222 

Proteids 224 

Milk  sugar 225 

Interpretation  of  results  226 


xii  CONTENTS. 

Preservatives  and  products  of  fermentation 228 

Formaldehyde  228 

Hydrogen  peroxide  230 

Salicylic  acid  and  salicylates  231 

Boric  acid  and  borates  232 

Fluorides  233 

Carbonates  and  bicarbonates 233 

Acidity 233 

Ammonia  234 

Reference  books 235 

ADDITIONAL  NOTES  AND  CORRECTIONS 237 

SUBJECT  I NDEX 238 


CHAPTER    I. 
Introduction. 

ULTIMATE   AND    PROXIMATE  ANALYSIS. 

Ultimate  organic  analysis  is  the  determination  of  the  elements 
composing  any  organic  substance.  Proximate  organic  analysis  is 
the  determination  of  the  compounds  present  in  a  mixture,  or  of  the 
radicals  present  in  a  compound. 

Both  ultimate  and  proximate  analyses  are  often  required  in  the 
examination  of  organic  materials.  In  the  case  of  a  complex  mix- 
ture, however,  proximate  analysis  is  frequently  directed  to  the 
determination  of  the  principal  groups  of  related  compounds  rather 
than  of  each  individual  compound  present. 

In  the  analysis  of  ordinary  animal  and  vegetable  substances  it  is 
usually  assumed  that  all  of  the  organic  matter  is  made  up  of  not 
more  than  six  elements  —  carbon,  hydrogen,  nitrogen,  oxygen, 
sulphur  and  phosphorus.  Other  elements  present  would  usually 
be  found  in  the  ash,  though  not  always  without  loss  as  will  be  ex- 
plained later. 

While  the  results  of  ultimate  analysis  are  often  calculated  to  the 
basis  of  dry,  ash-free  substance,  it  is  not  to  be  supposed  that  the 
ash  like  the  moisture  is  simply  mixed  with  the  organic  matter  of 
the  original  sample.  In  many  cases  the  inorganic  compounds 
found  in  the  ash  are  formed  during  combustion,  the  bases  having 
existed  in  combination  with  organic  acids  or  proteids  while  the 
acid  radicals  may  also  have  existed  in  organic  combination  or  may 
have  been  formed  by  the  oxidation  of  the  carbon,  sulphur  and 
phosphorus  of  the  organic  matter. 

The  preliminary  treatment  of  samples  and  the  most  generally 
applicable  methods  of  ultimate  analysis  including  the  preparation 
and  analysis  of  ash,  will  be  briefly  outlined  in  this  chapter.  In  the 
one  following,  those  methods  which  are  more  particularly  adapted 
to  the  determination  of  nitrogen,  sulphur  and  phosphorus  in  animal 
and  vegetable  substances  will  be  more  fully  described. 

PRELIMINARY   TREATMENT    OF    SAMPLES. 

Samples  received  and  analyzed  in  liquid  form  should  be  kept  in 
well-filled  tightly-stoppered  bottles  and  so  far  as  possible  in  a  cool 


2  ORGANIC   ANALYSIS. 

dark  place.  If  the  liquid  already  contains  an  antiseptic  or  is  rich 
in  any  such  preservative  substance  as  salt,  glycerol  or  alcohol,  or 
if  it  can  be  kept  at  a  sufficiently  low  temperature,  no  further  pre- 
cautions regarding  the  preservation  of  the  sample  will  be  required. 

When  there'is  danger  of  fermentation  the  sample  should  either 
be  sterilized  by  heat  or  preserved  by  freezing  or  by  the  addition 
of  an  antiseptic,  the  latter  method  being  usually  the  most  practi- 
cal. The  choice  of  antiseptic  will  depend  upon  the  nature  of  the 
sample  and  of  the  analysis  to  be  made.  Mercuric  chloride,  hydro- 
gen peroxide,  formaldehyde,  salicylic  acid,  chloroform  and  thymol 
are  among  the  most  useful  preservatives.  Formaldehyde  added  in 
the  proportion  of  0.2  to  0.3  c.c.  of  the  commercial  forty  per  cent, 
solution  to  each  100  c.c.  of  sample  is  especially  convenient  and 
efficient  but  cannot  be  used  in  all  cases  as  it  may  interfere  with 
some  of  the  tests  to  be  made  in  the  course  of  the  analysis. 

No  fixed  rules  can  be  given  for  the  preliminary  treatment  of 
solid  samples  but  the  following  directions  will  apply  in  most  cases. 

If  the  sample  is  moist,  dry  it  thoroughly  in  a  boiling  water  oven, 
allow  to  cool  in  the  air,  note  the  loss  in  weight  and  use  this  "  air- 
dry  "  sample  for  analysis.  Always  determine  moisture  in  the  air- 
dry  sample  so  that  the  analytical  results  can  be  calculated  to  the 
"  water-free  "  basis  if  desired. 

An  ordinary  drug  or  coffee  mill  *  is  usually  better  than  a  mor- 
tar for  grinding  the  air-dry  sample.  For  proximate  analysis  ot 
plant  or  animal  tissues  the  preliminary  grinding  should  theoret- 
ically be  so  thorough  as  to  break  all  of  the  cells.  In  most  cases 
it  is  found  sufficient  to  grind  until  the  sample  passes  a  sieve  hav- 
ing holes  one-half  millimeter  in  diameter. 

Substances  too  tough  or  sticky  to  be  ground  by  ordinary  means 
can  often  be  prepared  by  rasping  or  by  grinding  in  a  mortar  with 
a  known  proportion  of  sharp  sand  or  broken  glass. 

OUTLINE   OF   ULTIMATE   ORGANIC  ANALYSIS. 

Ultimate  organic  analysis  is  discussed  in  so  many  well-known 
laboratory  manuals  that  no  attempt  will  be  made  to  include  the 
subject  here.  For  comparison  with  the  methods  to  be  given  in 

*  For  laboratories  where  many  fibrous  samples  are  to  be  ground  the  Dreef  mill 
is  very  advantageous.  A  description  of  this  and  other  special  forms  of  grinding  ap- 
paratus will  be  found  in  Wiley's  Agricultural  Analysis,  Volume  III,  Part  First. 
(Easton,  Pa.,  1897). 


INTRODUCTION.  3 

the  next  chapter,  however,  the  most  generally  applicable  processes 
will  be  mentioned  with  references  to  some  of  the  more  complete  or 
recent  descriptions. 

In  the  determination  of  carbon  and  hydrogen  the  sample  is 
usually  burned  in  a  combustion  tube  in  a  current  of  air  or  oxygen, 
the  products  of  combustion  being  led  over  hotcopper  oxide  to  ensure 
complete  oxidation.  The  resulting  water  and  carbon  dioxide  are 
absorbed  respectively,  either  by  calcium  chloride  and  potassium 
hydroxide  or  by  sulphuric  acid  and  soda-lime.  This  method 
requires  modification  when  the  sample  contains  halogens,  light 
metals,  or  considerable  amounts  of  nitrogen  or  sulphur.  For  a 
discussion  of  such  modifications  as  well  as  for  the  essential  details 
of  the  process,  the  works  of  Benedict,*  Hans  Meyer  f  and  Lassar- 
Cohn  J  as  well  as  older  standard  works  should  be  consulted. 

For  the  determination  of  nitrogen  the  Dumas  method  alone  is 
applicable  to  all  organic  compounds.  The  substance  is  burned 
with  copper  oxide  and  the  products  of  combustion  passed  over  hot 
metallic  copper.  The  nitrogen  gas  thus  obtained  is  measured  and 
its  weight  calculated.  For  details  of  this  method  see  Gattermann,§ 
Hans  Meyer  (/.  <:.),  or  the  methods  of  the  Association  of  Official 
Agricultural  Chemists.  || 

For  the  determination  of  sulphur  and  phosphorus,  the  well- 
known  method  of  Carius  is  considered  the  most  generally  applic- 
able. The  substance  is  oxidized,  by  heating  with  fuming  nitric 
acid  in  a  sealed  tube  and  the  resulting  sulphuric  and  phosphoric 
acids  are  determined  by  the  usual  methods.  The  manipulation  of 
the  Carius  method  is  fully  described  by  Gattermann. 

Ultimate  analyses  can  also  be  made  by  burning  the  substance 
with  an  excess  of  oxygen  in  a  closed  chamber  such  as  the  bomb 
used  for  calorimetric  work  or  a  specially  constructed  small  autoclave. 
This  method,  proposed  by  Berthelot  If  and  developed  largely  by 
Hernpel,**  has  been  used  extensively  in  fuel  analyses  by  Lang- 

*  Elementary  Organic  Analysis,  Easton,  Pa.,  1900. 

f  Analyse  and  Konstitutionsermittlung  organischer  Verbindungen,  Berlin,  1903. 

J  Arbeitsmethoden  fiir  Organisch-chemische  Laboratorien    [3  Aufl.],  Hamburg, 
1903. 

§  Practical  Methods  of  Organic  Chemistry,  New  York,  1901. 

||  Bulletin  46,  Bureau  of  Chemistry,  U.  S.  Department  of  Agriculture. 

^Compl.  rend.,  1892,  114,  318;  1899,  129,  IOO2  ;  Ztschr.  anal.  Chem.,  1901. 
40,  124. 

**  Ber.  deut.  c/iem.  Ges.,  1897,  30,  202. 


4  ORGANIC   ANALYSIS. 

bein.*     Its  application  to  the  determination  of  sulphur  is  given  in 
the  next  chapter. 

PREPARATION    AND    ANALYSIS    OF    ASH. 
PREPARATION  OF  ASH. 

Incineration  of  ordinary  substances  is  best  accomplished  in  a  flat 
platinum  dish  in  a  muffle.  The  substance  should  first  be  carefully 
charred  at  the  front  of  the  muffle,  then  burned  until  white  or  very 
light  gray  at  a  temperature  of  500°  to  6oo°.t  If  the  temperature 
cannot  be  accurately  known,  the  operation  should  be  carefully 
watched  and  the  dish  never  heated  above  a  very  low  redness. 
Higher  temperatures  not  only  increase  the  loss  of  chlorides  but 
may  cause  the  ash  to  fuse  over  some  of  the  carbon  and  thus  greatly 
retard  its  combustion.  If  the  substance  is  rich  in  salts  of  the 
alkalies,  char  it  gently,  extract  with  warm  water,  pour  off  the 
aqueous  extract  and  set  aside  while  burning  the  carbonaceous  resi- 
due to  whiteness,  then  return  the  aqueous  extract  to  the  platinum 
dish,  evaporate  to  dry  ness,  and  ignite  gently  as  already  directed. 
Animal  substances,  seeds,  and  other  samples  rich  in  phosphates 
should  be  extracted  with  acetic  acid  instead  of  with  water  alone. 

Various  forms  of  special  apparatus  for  the  preparation  of  ash 
have  been  described,  those  of  Shuttle  worth,  J  Tucker  §  and  Wis- 
licenus  ||  being  probably  the  best  known. 

In  order  to  avoid  discrepancies  due  to  the  absorption  of  moisture 
from  the  air,  the  ash  may  be  thoroughly  mixed  while  still  warm 
and  transferred  at  once  to  a  tightly  stoppered  weighing  bottle. 
The  ash  being  then  perfectly  dry,  no  determination  of  moisture  is 
necessary  and  the  portions  required  for  analysis  may  be  weighed 
out  by  difference. 

METHODS  OF  ASH  ANALYSIS. 

Since  almost  any  of  the  elements  found  in  soil  or  water  may  also 
be  found  in  ash,  no  attempt  to  outline  a  complete  scheme  of 

*  Ztschr.  angew.  Chem.,  1900,  1227,  1259;   1901,  516. 

t  In  a  series  of  experiments  by  Thompson  and  the  writer  in  which  portions  of  dried 
milk  were  incinerated  at  known  temperatures  in  an  electrically  heated  muffle,  it  was 
found  that  increase  of  temperature  from  450°  to  600°  had  no  appreciable  influence  upon 
the  amount  of  chlorine  recovered  in  the  ash,  but  that  a  loss  occurred  at  about  650°.  For 
other  results  of  this  study  see  the  chapter  on  milk  analysis. 

J  Dissertation,  Goettingen,  1899.     Journ.  Landw.,  1899,  47,  173. 

$  Ber.  deut.  chem.  Ges.y  1899,  32,  2583. 

||  Ztschr.  anal.  Chem.,  1901,  40,  441. 


INTRODUCTION.  5 

analysis  would  be  of  much  practical  use,  especially  since  the 
amount  of  ash  available  is  generally  so  small  that  the  analysis  must 
be  limited  to  the  determination  of  the  more  important  constituents. 
The  determinations  most  often  required  may  be  carried  out  in 
accordance  with  the  plan  adopted  by  the  Association  of  Official 
Agricultural  Chemists  (/.  <:.).  Methods  of  ash  analysis  are  also 
described  in  Fresenius'  Quantitative  Analysis  and  in  a  paper  by 
Tollens  in  the  Experiment  Station  Record,  Volume  XIII,  pages  207- 
220.  For  detailed  descriptions  of  the  separations  and  determina- 
tions required,  text-books  on  inorganic  quantitative  analysis  * 
should  be  consulted.  Small  amounts  of  iron  may  conveniently  be 
estimated  by  colorimetric  methods. f 

INTERPRETATION  OF  ASH  ANALYSIS. 

Chlorine.  —  Even  when  the  incineration  has  been  carried  on  care- 
fully at  temperatures  considerably  below  those  at  which  the  chlo- 
rides begin  to  volatilize,  the  whole  of  the  chlorine  of  the  original 
substance  is  rarely  recovered  in  the  ash.J  Apparently  this  loss  of 
chlorine  is  due  to  the  formation  of  acid  products  in  the  early  stages 
of  ignition  of  the  organic  matter,  when  the  conditions  approach 
those  of  dry  distillation.  When  the  relative  amount  of  organic 
matter  is  large  these  acid  products  are  formed  in  such  quantity  as 
to  expel  a  considerable  part  of  the  chlorine  from  its  combination 
with  the  alkalies.  The  alkali  thus  retained  in  combination  with 
organic  matter  is  converted  to  carbonate  by  further  ignition,  and 
this  in  turn  may  be  acted  upon  by  some  stronger  acid  formed 
during  the  combustion.  The  loss  of  chlorine  increases  with  the 
proportion  of  organic  matter  to  chloride.  According  to  Davis  §  it 
may  be  prevented  by  the  addition  of  sodium  carbonate  to  the  ex- 
tent of  5  per  cent,  of  the  weight  of  organic  matter  present,  but  this 
of  course  is  not  permissible  in  the  preparation  of  ash  for  general 

*  Among  the  more  recent  are  —  Classen  :  Ausgewahlte  Methoden  der  Analytischen 
Chemie,  Braunschweig,  1901-1903.  Treadwell :  Analytical  Chemistry,  Vol.  II 
(Quantitative  Analysis),  New  York,  1904.  Miller:  Quantitative  Analysis  for  Mining 
Engineers,  New  York,  1904. 

|  Thompson:  Journ.  Chem.  Soc.,  1885,  47,  493.  Borntrager :  Chem.  Ztg.,  1896, 
20,  398.  Richards  and  Woodman  :  Air,  Water  and  Food,  New  York,  1904.  Pul- 
sifer  :  Journ,  Amer.  Chem.  Soc.,  1904,  26,  967. 

J  For  a  very  full  discussion  of  the  loss  of  chlorine  in  the  preparation  of  ash,  includ- 
ing references  to  earlier  work  see  Adlerskron  :  Ztschr.  anal.  Chem.,  1873,  I2>  39°- 

$  Journ.  Soc.  Chem.  Ind.,  1901,  20,  98. 


6  ORGANIC   ANALYSIS. 

analysis.  Shuttleworth  *  recommends  calcium  acetate  as  being 
efficient  in  preventing  loss  of  chlorine  and  especially  convenient  in 
the  incineration  of  siliceous  substances  such  as  straw. 

Carbonates.  —  Carbonates  in  ash  are  not  derived  necessarily  from 
salts  of  carbonic  or  organic  acids  existing  in  the  original  sub- 
stance. The  bases  found  as  carbonates  in  the  ash  may  have  been 
combined  with  proteids  or  may  have  been  derived  from  inorganic 
salts  decomposed  by  heating  in  contact  with  the  large  excess  of 
organic  matter,  as  just  mentioned  in  the  case  of  alkaline  chlorides. 
On  the  other  hand,  carbonates  originally  present  or  formed  during 
ignition  may  be  changed  to  sulphates  or  phosphates  by  the  action  of 
acids  resulting  from  the  oxidation  of  organic  sulphur  or  phosphorus. 

Sulphates.  —  The  amount  of  sulphate  found  in  the  ash  bears  little 
relation  either  to  the  total  sulphur,  or  to  that  existing  as  sulphate., 
in  the  original  substance.  Depending  upon  the  other  constituents 
present  and  the  conditions  of  incineration,  sulphates  may  be  reduced 
or  converted  to  phosphates,  or,  on  the  other  hand,  the  organic  sul- 
phur may  be  largely  oxidized  and  retained  as  sulphate.  Very 
rarely  in  the  combustion  of  natural  products  does  all  of  the  sulphur 
remain  in  the  ash.  More  frequently  only  a  small  fraction  of  it  is 
thus  retained. 

Phosphates.  —  The  phosphates  of  the  ash  usually  represent  the 
whole  of  the  pre-existing  inorganic  phosphates  and,  in  the  case  of 
natural  substances,  usually  much  of  the  phosphorus  which  was 
present  in  organic  combination.  Since  the  latter  exists  mainly,  if 
not  entirely,  as  organic  phosphates  or  substituted  phosphoric  acid, 
it  is  largely  burned  to  free  orthophosphoric  acid  and  thus  remains 
as  mineral  phosphate  in  the  ash.  With  many  substances,  practi- 
cally all  of  the  phosphorus  is  thus  retained  as  phosphate  by  the 
ash,  but  it  cannot  be  assumed  that  such  will  be  the  case  whenever 
there  are  bases  enough  to  combine  with  all  of  the  phosphoric  acid. 
Thus  there  is  some  loss  of  phosphorus  in  burning  the  dried  residue 
of  milk  at  600°,  although  the  ash  thus  obtained  contains  a  consid- 
erable proportion  of  chlorides.  When  the  phosphoric  acid  formed 
is  more  than  sufficient  to  saturate  the  bases  present,  the  excess  of 
acid  is  very  slowly  volatilized.  In  such  cases  the  ash  is  apt  to  fuse 
over  the  last  portions  of  unburned  carbon,  interfering  both  with 
the  determination  or  preparation  of  ash  and  with  the  determination 
of  carbon  in  the  elementary  analysis. 

*  Dissertation,  Goettingen,  1899. 


INTRODUCTION.  7 

Bases.  —  Some  of  the  heavy  metals,  if  present,  may  be  reduced 
and  volatilized  during  the  combustion  of  the  organic  matter. 
There  is  danger  of  mechanical  loss  of  ash  if  combustion  is  carried 
on  rapidly  or  in  too  strong  a  current  of  air.  A  sufficient  circula- 
tion of  air  will  be  secured  by  inclining  slightly  the  muffle  in  which 
the  substance  is  burned.  The  volatilization  of  alkaline  chlorides 
as  such  at  temperatures  of  650°  or  over  has  already  been  mentioned. 

When  small  amounts  of  metallic  bases  are  to  be  determined  it  is 
usually  advantageous  and  often  necessary  to  destroy  the  organic 
matter,  in  part  at  least,  by  boiling  with  acids  instead  of  by  burning 
in  the  air.* 

*  Neumann:  Ztschr.  physiol.  Chem.,  1902-3,  37,  115.     Meilliere  :  Journ.  Pharm. 
.y  1902,  [6]  15,  97.     Munson  :    U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bull.  65,  p.  53. 


CHAPTER    II. 
Nitrogen,  Sulphur  and  Phosphorus. 

THE    DETERMINATION   OF    NITROGEN. 

The  method  of  Dumas  as  outlined  in  the  preceding  chapter  has 
the  advantage  of  being  applicable  to  all  classes  of  nitrogen  com- 
pounds. In  the  great  majority  of  cases,  however,  it  is  equally  accu- 
rate and  much  more  convenient  to  use  one  of  the  methods  based 
upon  the  conversion  of  nitrogen  to  ammonia  and  the  determination 
of  the  latter  by  titration. 

Before  the  introduction  of  the  Kjeldahl  process,  the  soda-lime 
method  of  Will  and  Varrentrap  was  commonly  used.  In  this 
method  the  finely  ground  substance  is  mixed  with  a  large  excess 
of  soda-lime  and  heated  in  a  combustion  tube,  the  ammonia  given 
off  being  absorbed  in  standard  acid.  In  order  to  ensure  the  reduc- 
tion of  nitro-compounds  or  nitrates,  this  method  was  modified  by 
the  introduction  of  stannous  sulphide  (Goldberg) ;  of  sodium 
formate  and  sodium  thiosulphate  (Arnold);  or  of  sodium  thio- 
sulphate  and  a  mixture  of  equal  parts  sulphur  and  powdered  sugar 
or  charcoal  (Ruffle).  The  soda-lime  method  has  now  been  very 
generally  superseded  by  the  various  modifications  of  the  Kjeldahl 
process. 

THE  KJELDAHL  METHOD. 

In  this  process  the  substance  is  decomposed  by  heating  with 
strong  sulphuric  acid,  usually  with  the  addition  of  some  reagent 
which  assists  the  decomposition  either  by  raising  the  boiling  point 
or  by  acting  as  a  carrier  of  oxygen.  When  decomposition  is  com- 
plete the  nitrogen  remains  as  ammonium  sulphate  in  the  sulphuric 
stcid,  the  carbon  and  hydrogen  of  the  substance  having  been  oxi- 
dized and  the  products  of  oxidation  boiled  out  of  the  solution.  The 
oxidation  takes  place  partly  at  the  expense  of  the  sulphuric  acid 
so  that  a  considerable  evolution  of  sulphur  dioxide  occurs,  especially 
in  the  earlier  stages  of  the  process.  After  the  completion  of  the 
digestion,  the  ammonia  is  liberated  by  means  of  fixed  alkali  and 
determined  by  distilling  into  standard  acid.  In  the  various  modi- 
fications of  the  process,  different  reagents  or  combinations  of 
reagents  are  used  to  hasten  the  decomposition  of  the  organic 
matter  by  the  sulphuric  acid. 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.          9 

Kjeldahl  originally  directed  *  that  the  substance  be  heated  with 
sulphuric  acid,  with  or  without  the  addition  of  phosphoric  anhydride, 
until  a  clear  solution  is  obtained ;  then  potassium  permanganate 
added  in  small  portions  to  the  hot  solution  until  it  remains  perma- 
nently colored,  the  permanganate  being  added  very  cautiously  on 
account  of  the  danger  of  a  loss  of  nitrogen  if  the  reaction  becomes 
too  vigorous. 

Willfarth  |  introduced  the  use  of  mercuric  oxide  to  facilitate 
the  action  of  the  sulphuric  acid,  and  stated  that  if  the  solution  be 
boiled  until  colorless  neither  phosphoric  anhydride  nor  potassium 
permanganate  need  be  used.  In  his  earlier  experiments  Willfarth 
used  copper  instead  of  mercury.  Arnold  J  used  both  mercury  and 
copper  in  addition  to  phosphoric  anhydride. 

Gunning  §  used  for  the  digestion  a  simple  mixture  of  sulphuric 
acid  with  one-third  to  one-half  its  weight  of  potassium  sulphate. 
Arnold  and  Wedemeyer  ||  modified  the  Gunning  process  by  the 
use  of  mercury  and  copper  in  addition  to  the  potassium  sulphate. 
By  this  modification  the  time  required  to  decompose  the  organic 
matter  was  greatly  reduced  and  good  results  were  obtained  with  a 
number  of  alkaloids  and  other  compounds  which  had  not  readily 
yielded  the  whole  of  their  nitrogen  when  treated  by  the  methods 
previously  used.  Independently  Dyer^f  obtained  equally  good 
results  on  a  wide  range  of  organic  compounds  by  the  use  of  mer- 
cury and  potassium  sulphate  without  copper. 

The  official  agricultural  chemists  authorize**  the  Kjeldahl- 
Willfarth  and  the  Gunning  methods  for  the  analysis  of  foods, 
spices  other  than  peppers,  and  fertilizers  not  containing  nitrates. 
For  peppers,  to  secure  complete  ammonification  of  the  alkaloidal 
nitrogen,  the  Arnold-Wedemeyer  modification  of  the  Gunning 
method  was  provisonally  adopted  in  1902.  In  the  experience  of 
this  laboratory,  the  Dyer  modification  has  been  found  more  rapid 
and  slightly  more  accurate  than  either  the  Kjeldahl-Willfarth  or 
Gunning  method  and  fully  as  efficient  in  the  case  of  alkaloids 
as  is  the  Arnold-Wedemeyer  method,  while  it  has  the  advantage 

*  Ztschr.  anal,    Chem.,  1883,  22,  366. 

f  Chem.  Centrbl.  1885  [3]  16,  17,  113. 

\Archiv.  der  Pharm.  [3],  24,  785  ;  Ztschr.  anal.  Chem.,  1887,  26,  249. 

§  Ztschr.  anal.  Chem.,  1889,  28,  188. 

||  Ztschr.  anal.  Chem.,  1892,  31,  525. 

^Journ.  Chem.  Soc.,  1895,  67,  8li. 

**  Bulletins  46  and  65,  Bureau  of  Chemistry,  U.  S.  Dept.  Agriculture. 


io  ORGANIC   ANALYSIS. 

over  the  latter  of  requiring  one  less  reagent  and  of  yielding  a 
colorless  solution. 

Applicability  'of  the  Kjeldahl  Method. — The  Gunning- Arnold-Dyer 
modification,  which  for  the  reasons  just  mentioned  is  recommended 
for  general  use,  is  applicable  to  all  classes  of  animal  and  vegetable 
substances,  including  such  difficultly  decomposable  bases  as  betaine 
and  the  pyridine  and  chinoline  alkaloids,  and  to  cyanides,  ferrocy- 
anides  and  ferricyanides.  It  has  also  been  tested  *  with  good 
results  on  many  other  compounds,  including  acetanilid,  sulphanilic 
acid,  orthobenzoic  sulphinid,  amidobenzoic  acid,  benzamid,  diami- 
dophenol,  naphthylamine,  diphenylamine,  diphenylthiourea,  nitro- 
so-dimethylaniline,  indigotin,  pyridine,  and  oxyphenyl  methylpyri- 
midine. 

Jodlbauer's  modification,!  devised  for  the  determination  of  nitro- 
gen in  nitrates,  was  found  by  Dyer  to  be  applicable  to  nitro-com- 
pounds,  to  azo-,  hydrazo-  and  amidoazo-benzene,  to  carbazol  and, 
with  the  addition  of  I  or  2  grams  of  sugar,  to  hydroxylamine  and 
oximes.  Dyer  did  not  obtain  the  whole  of  the  nitrogen  of  hydra- 
zine  derivatives  but  Dafert  \  and  Milbauer  §  have  published  modi- 
fications which  are  said  to  give  accurate  results  with  this  class  of 
compounds. 

GUNNING-ARNOLD-DYER  MODIFICATION. 

Reagents. —  Pure  concentrated  sulphuric  acid.  Mercury.  Pure 
potassium  sulphate.  Potassium  sulphide  solution,  40  grams  per 
liter.  Saturated  solution  of  sodium  hydroxide  (commercial). 
Granulated  zinc  or  pumice  stone.  Paraffin.  Solution  of  methyl- 
orange,  congo  red,  cochineal,  lacmoid,  or  any  other  indicator  suit- 
able for  titration  in  the  presence  of  ammonium  salts.  Standard 
solutions  of  acid  and  alkali,  preferably  one-fifth  or  one-tenth  normal. 

Determination.  —  Weigh  0.5  to  5.0  grams  sample,  or  so  much  as 
will  probably  yield  from  50  to  75  milligrams  of  ammonia,  and 
transfer  to  a  pearshaped  Kjeldahl  flask  of  550  to  750  c.c.  capacity. 
Add  20  to  25  c.c.  concentrated  sulphuric  acid  and  about  0.7  gram 
of  mercury.  Place  the  flask  in  an  inclined  position  and  heat  gently 
until  the  first  vigorous  frothing  ceases,  then  raise  the  heat  grad- 

*  In  some  cases  without  the  use  of  mercury.     See  in  addition  to  the  papers  already 
cited,  that  of  Gibson  in  the  Journal  of  the  American  Chemical  Society,  1904,  26,  105. 
f  Chem.  Centrbl.,  1886,  3,  17,  433. 
\Land-w.    Versuchs-Sta.,  1887,  34,  311. 
§Ztschr.  anal.  Chem.,  1903,  42,  725. 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.         1 1 

ually  until  the  liquid  boils  ;  remove  the  flame  for  a  few  minutes, 
add  10  to  12  grams  of  potassium  sulphate  and  boil.  If  the  liquid 
is  kept  actually' boiling,  it  will  usually  be  clear  and  colorless  within 
30  minutes  after  the  addition  of  the  sulphate.  Continue  the  boil- 
ing for  at  least  30  minutes  after  the  solution  becomes  colorless  or 
in  any  case  for  one  hour  from  the  time  the  potassium  sulphate 
is  added.  If  the  sample  contains  alkaloids,  the  boiling  should 
be  continued  for  at  least  three  hours  in  all  and  not  less  than  two 
hours  after  the  solution  is  colorless.  When  the  digestion  is  finished, 
allow  the  flask  to  cool  for  10  to  15  minutes  or  to  4O°-6o°  (if  allowed 
to  become  thoroughly  cold  the  solution  solidifies) ;  then  dilute  care- 
fully with  150  to  200  c.c.  of  water;  allow  to  cool,  add  25  c.c.  potas- 
sium sulphide  solution,  mix  well  and  then  add  75  to  100  c.c.  (or 
enough  to  make  the  reaction  strongly  alkaline*)  of  a  cold  saturated 
solution  of  sodium  hydroxide,  pouring  it  carefully  down  the  side 
of  the  flask,  so  that  it  does-  not  mix  immediately  with  the  acid 
solution.  Add  a  few  pieces  of  granulated  zinc  or  pumice  stone  to 
prevent  bumping,  and  a  piece  of  paraffin  the  size  of  a  pea  to 
diminish  frothing;  connect  the  flask  (preferably  by  means  of  a 
Hopkins'  distilling  head)  with  a  condenser,  the  delivery  tube  of 
which  dips  into  50  c.c.  of  N/io  sulphuric  or  hydrochloric  acid  or 
its  equivalent  in  the  receiver ;  mix  the  contents  of  the  flask  by 
shaking  and  distil  until  about  one-half  of  the  liquid  has  passed  into 
the  receiver.  Titrate  the  excess  of  acid  in  the  receiver  by  means 
of  standard  alkali,  using  one  of  the  indicators  mentioned  above. 

The  reagents  used  should  be  tested  by  making  a  blank  determi- 
nation with  pure  sugar  or  cellulose,  carrying  out  all  operations  in 
exactly  the  same  way  as  in  a  regular  analysis. 

Notes.  —  If  in  transferring  the  substance  to  the  flask  any  par- 
ticles or  drops  should  lodge  in  the  neck,  they  can  be  washed  down 
by  the  sulphuric  acid  subsequently  added.  Dry  samples  can 
usually  be  weighed  on  a  watchglass  and  brushed  into  the  flask 
through  a  funnel  having  a  wide  stem  which  has  been  cut  down  to 
a  length  of  about  one  cm.,  or  transferred  by  means  of  a  narrow 
strip  of  glazed  paper.  It  is  often  more  convenient  to  weigh  the 
sample  on,  or  transfer  to,  a  small  piece  of  pure  filter  paper,  fold  the 
latter  loosely  over  the  weighed  portion  and  introduce  the  whole 
into  the  flask  in  such  a  way  that  the  sample  can  be  easily  shaken 

*Corallin  (rosolic  acid)  may  be  used  as  indicator  to  show  that  the  contents  of  the 
flask  are  alkaline  before  beginning  the  distillation. 


12  ORGANIC   ANALYSIS. 

free  from  the  paper.  The  amount  of  cellulose  thus  introduced 
with  the  sample  does  not  materially  prolong  the  time  required  for 
digestion,  while  its  reducing  effect  may  aid  in  the  ammonification 
of  any  firmly  bound  nitrogen  present.  Time  should  be  allowed 
for  thorough  wetting  of  the  sample  by  the  sulphuric  acid  before 
heat  is  applied.  During  digestion  a  small  funnel  or  balloon  stop- 
per may  be  placed  loosely  in  the  mouth  of  the  flask  to  retard  the 
evaporation  of  acid  and  guard  against  mechanical  loss.  The  flask 
may  rest  upon  a  wire  gauze,  an  iron  plate  or  a  piece  of  asbestos 
having  a  circular  hole  about  4  cm.  in  diameter  which  permits  the 
free  flame  of  the  Bunsen  burner  to  play  upon  the  flask  below  the 
surface  of  the  boiling  liquid.  The  digestion  should  be  conducted 
in  a  well  ventilated  hood  where  the  air  is  free  from  any  consider- 
able amount  of  ammonia.  In  order  to  hasten  the  decomposition 
of  organic  matter,  Dakin  *  and  Milbauer  (/.  r.)  recommend  the  use 
of  potassium  persulphate,  while  Krugerf  suggests  the  addition  of 
potassium  dichromate  in  excess  of  the  amount  necessary  to  oxidize 
all  carbon  and  hydrogen  present.  The  method  here  described  is, 
however,  sufficiently  rapid  and  more  convenient  for  ordinary  work. 
The  precipitation  of  mercury  as  sulphide  before  rendering  the 
solution  alkaline  is  to  prevent  the  formation  of  mercur-ammonium 
compounds  which  do  not  readily  yield  their  ammonia  on  boiling 
with  caustic  alkali.  An  ordinary  Liebig  condenser  can  be  used  in 
this  distillation,  though  block  tin  tubing  is  generally  preferred  as 
being  more  resistant  to  the  action  of  steam  and  ammonia.  Unless 
the  condenser  tube  is  straight  and  nearly  vertical,  it  is  safer  to 
rinse  it  out  with  a  little  cold  distilled  water  at  the  end  of  the  dis- 
tillation and  add  the  rinsings  to  the  distillate.  The  distillation  as 
usually  carried  out  requires  from  forty  minutes  to  one  hour.  Only 
about  one  third  of  this  time  is  actually  required  to  expel  the  am- 
monia from  the  boiling  solution  and  if  it  is  desired  to  hasten  the 
operation,  the  steam  can  be  conducted  through  tin  tubing  without 
condensation  into  a  cooled  receiver ;  or,  as  suggested  by  Benedict.^ 
the  water  which  cools  the  condenser  may  be  drawn  off  after  the 
solution  has  been  boiling  15  minutes,  and  the  boiling  continued 
until  enough  steam  has  passed  through  to  carry  all  of  the  ammonia 
from  the  condenser  tube  into  the  receiver.  Fixed  alkali  is  prefer- 

*  Journ.  Soc.  Chem.  Ind.,  1902,  21,  848. 
t  Ber.  deut.  chem.  Ges.,  1894,  27,  609. 
\Journ.  Amer.  Chem,  Soc.,  1900,  22,  259. 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.         13 

able  to  ammonia  for  the  final  titration  since  the  latter  reagent  is 
liable  to  lose  strength  during  use,  probably  through  evaporation  of 
ammonia  from  the  tip  of  the  burette  and  from  the  falling  drop. 

The  reasons  for  preferring  the  method  as  above  described  to  the 
original  Kjeldahl-Willfarth  or  Gunning  method  have  been  briefly 
outlined  above.  If  either  of  the  latter  methods  is  used,  the 
same  details  of  manipulation  may  be  followed  except  that  the 
digestion  should  be  continued  for  at  least  two  hours  after  the  solu- 
tion becomes  colorless  and  not  less  than  three  hours  in  all.  Even 
with  this  longer  time  of  boiling  the  results  are  often  slightly  low. 
For  the  results  of  a  comparative  study  of  the  Kjeldahl-Willfarth, 
the  Gunning,  and  the  above  described  method  with  special  refer- 
ence to  the  time  of  boiling  required  see  the  Journal  of  the  Ameri- 
can Chemical  Society  for  April  and  November,  1904. 

METHOD  FOR  NITRATES  AND  NITRO- COMPOUNDS. 

The  loss  of  nitric  acid  which  might  otherwise  occur  when  sul- 
phuric acid  is  poured  upon  a  sample  containing  nitrates,  is  avoided 
by  having  present  some  substance  which  is  very  easily  nitrated, 
such  as  salicylic  acid,  benzoic  acid  or  phenol.  The  nitrogen  having 
been  thus  fixed  as  a  nitro-compound,  the  latter  may  be  reduced  by 
the  addition  of  zinc  dust  (J  odlbauer)  or  sodium  thiosulphate  (Forster) 
after  which  the  determination  is  carried  out  in  the  usual  manner. 

Reagents.  —  Sulphuric-salicylic  acid,  prepared  in  advance  by  dis- 
solving salicylic  acid  in  pure  concentrated  sulphuric  in  the  propor- 
tion of  2  grams  of  the  former  to  30  c.c.  of  the  latter.  Zinc  dust,  an 
impalpable  powder  ;  or  crystallized  sodium  thiosulphate.  Other 
reagents  as  for  the  method  above  described. 

Determination. —  To  the  weighed  portion  of  substance  in  a 
Kjeldahl  flask,  add  30  c.c.  of  the  sulphuric-salicylic  acid  solution 
and  shake  until  thoroughly  mixed ;  then  add  5  grams  crystallized 
sodium  thiosulphate,  or  add  gradually  with  constant  shaking  2 
grams  of  zinc  dust;  warm  gently  until  frothing  subsides  and  then 
boil  until  thick  white  fumes  cease  to  be  given  off.  Allow  to  cool 
somewhat,  add  mercury  and  potassium  sulphate  and  complete  the 
process  as  described  above.  Or  the  potassium  sulphate  may  be 
omitted  and  the  oxidation  completed  by  means  of  permanganate 
as  in  the  'modified  Kjeldahl'  method  of  the  official  agricultural 
chemists. 

Notes. —  Thiosulphate    is    the  more  convenient  reducing  agent 


14  ORGANIC   ANALYSIS. 

and  can  be  safely  used  if  the  amount  of  nitric  nitrogen  is  small. 
Zinc  dust  appears  to  be  a  safer  reagent  for  samples  rich  in  nitrates. 
In  the  presence  of  considerable  amounts  of  chloride  or  of  ammo- 
mium  salts  there  is  danger  of  a  loss  of  nitrogen  as  oxide  when  the 
sample  is  treated  with  the  sulphuric-salicylic  acid  mixture.  In 
such  cases  the  latter  mixture  should  be  thoroughly  cooled  before 
using  and  then  poured  as  quickly  as  possible  over  the  sample  and 
the  flask  allowed  to  stand  with  occasional  shaking  for  at  least  half 
an  hour  before  the  reducing  agent  is  added.  Ten  minutes  should 
be  allowed  for  the  reduction  to  take  place  before  the  solution  is 
heated.  With  these  precautions  the  loss  of  nitrogen  will  be  re- 
duced to  an  amount  too  small  to  be  appreciable  in  ordinary  work. 

THE    DETERMINATION    OF    SULPHUR. 

In  the  various  methods  employed  for  the  determination  of  sul- 
phur, organic  matter  is  destroyed  by  oxidation  and  the  resulting 
sulphuric  acid  is  precipitated  and  weighed  as  barium  sulphate  in 
the  usual  way.  The  chief  difficulty  lies  in  securing  complete 
oxidation  without  loss,  since  many  substances  contain  sulphur  in 
forms  from  which  it  is  easily  liberated  as  volatile  compound. 

COMPARATIVE  OUTLINE   OF  METHODS. 

The  principal  methods  of  bringing  about  the  oxidation  of  the 
organic  matter  are  : 

1.  By  heating  with  nitric  acid  or  an  oxidizing  acid  mixture. 

2.  By  burning  in  a  combustion  tube  in  a  current  of  air,  oxygen 
or  some  oxidizing  vapor. 

3.  By  heating  in  the  presence  of  alkali  and  completing  the  re- 
action by  the  addition  of  an  oxidizing  agent. 

4.  By  combustion  in  oxygen  in  a  closed  vessel. 

Since  the  choice  of  a  method  depends  on  the  nature  of  the 
sample  to  be  examined  and  the  apparatus  at  hand,  no  fixed  rules 
can  be  laid  down  for  this  purpose.  Several  of  the  more  important 
methods  are  therefore  outlined  and  three  methods  which  are 
especially  useful  for  plant  and  animal  substances  are  g-iven  in  full. 

Of  methods  of  the  first  group,  that  of  Carius,  which  has  already 
been  mentioned  in  the  previous  chapter,  is  the  most  important.  It 
is  applicable  to  volatile  as  well  as  to  non-volatile  substances,  but  is 
somewhat  troublesome  and  tedious,  and  determinations  are  fre- 
quently lost  through  breaking  of  the  heated  tubes,  especially  when 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.         15 

much  gas  is  produced  by  the  oxidation  of  the  carbon  of  the  sample. 
For  this  reason  only  a  small  amount  of  any  highly  carbonaceous 
substance  can  be  taken  for  analysis  and  often  the  amount  of  sul- 
phuric acid  obtained  is  too  small  for  satisfactory  determination. 

Several  methods  belonging  to  the  second  group  have  been  pro- 
posed. Among  the  more  important  are:  (a)  The  method  of  Bru- 
gelmann,*  in  which  the  substance  is  burned  in  a  current  of  oxygen, 
complete  oxidation  being  insured  by  passing  the  mixture  of  gases 
over  hot  platinum,  and  absorbing  the  sulphuric  acid  in  a  column 
of  soda-lime.  If  the  substance  is  poor  in  sulphur  several  portions 
can  be  burned  before  dissolving  out  the  lime  and  determining  the 
sulphuric  acid,  (b]  Mixter's  modification!  of  Sauer's  method.  This 
consists  in  burning  the  substance  in  a  current  of  oxygen  and  pass- 
ing the  products  of  combustion  into  bromine  water,  (c)  Classen's  J 
method  of  burning  in  a  current  of  nitric  oxide,  (d)  Barlow's 
method  §  in  which  the  substance  is  burned  in  a  current  of  oxygen 
in  a  specially  constructed  tube  and  complete  oxidation  is  insured 
by  means  of  a  second  current  of  oxygen  introduced  laterally  at  a 
point  between  the  boat  and  the  absorbing  column.  Methods  of 
this  type,  carefully  carried  out,  ensure  complete  oxidation  and  sev- 
eral portions  of  a  sample  may  be  burned  in  succession  so  as  to 
secure  a  larger  amount  of  sulphuric  acid  for  determination.  They 
are,  however,  less  rapid  than  the  methods  described  below,  which 
are  usually  quite  as  accurate. 

Liebig  destroyed  organic  matter  and  converted  sulphur  to  sul- 
phate by  heating  with  a  mixture  of  caustic  alkali  and  alkaline 
nitrate  and  methods  of  this  type  have  been  very  generally  used  for 
plant  and  animal  materials  and  other  non-volatile  substances.  Of 
the  many  modifications  of  Liebig's  method,  the  use  of  sodium  per- 
oxide as  an  oxidizing  agent  is  most  important.  The  peroxide  may 
be  used  in  connection  with  hydroxide  as  in  Osborne's  method,  or 
the  sample  may  be  mixed  at  once  with  an  excess  of  peroxide  and 
ignited  in  a  closed  crucible  ||  or  in  the  combustion  chamber  of  the 
Parr  calorimeter.^ 

*  Ztschr.  anal.  Chem.,  1876,  15,  I,  175;   1877,  16,  I. 

|  Amer.  Chem.  Journ.,  1880,  2,  396. 

\Ber.  deut.  chem.  Ges.,  1886,  19,  1910;   1887,  20,  3065. 

$  Journ.  Amer.  Chem.  Soc.,  1904,  26,  341. 

||  Sundstrom  :  Journ.  Amer.  Chem.  Soc.,  1903,  25,  184.  Pennock  and  Morton: 
Ibid.,  25,  1265. 

fl  Parr  :  Journ.  Amer.  Chem.  Soc.,  1900,  22,  646;  1904,  26,  1139.  Konek  : 
Ztschr.  angeiv.  Chem.,  1903,  516.  Leclerc  and  Dubois :  Journ.  Amer.  Chem.  Soc., 
1904,  26,  1108. 


16  ORGANIC   ANALYSIS. 

Berthelot,  in  proposing  the  use  of  the  oxygen  calorimeter  for  the 
elementary  analysis  of  organic  materials,  *  stated  that  on  combus- 
tion with  25  atmospheres  of  oxygen  the  sulphur  was  completely 
oxidized  and  in  the  presence  of  moisture  could  be  quantitatively 
recovered  as  sulphuric  acid.  This  method  has  been  found  both 
convenient  and  accurate.  Complete  oxidation  takes  place  instan- 
taneously, and  since  the  combustion  chamber  is  hermetically  sealed 
there  is  no  possibility  of  a  loss  of  volatile  compounds.  As  noted 
in  the  preceding  chapter,  a  small  autoclave  may  be  used  for  the 
combustion  if  a  bomb  calorimeter  is  not  available.  According  to 
Hempel,t  the  sample  can  be  burned  in  a  large  glass  bottle  filled 
with  oxygen  at  atmospheric  pressure. 

The  nature  and  number  of  the  samples  to  be  analyzed  will  usu- 
ally determine  which  of  the  above  methods  is  to  be  preferred.  For 
the  majority  of  cases  either  Liebig's  or  Osborne's  method,  or  if  the 
facilities  are  available,  Berthelot's  method  of  combustion  in  com- 
pressed oxygen  may  be  recommended. 

LIEBIG'S  ALKALI  METHOD. 

In  a  large  silver  or  nickel  crucible  mix  about  8  grams  potassium 
hydroxide  and  about  I  gram  potassium  nitrate.  Fuse  and  stir 
with  a  silver  or  nickel  spatula.  Allow  to  cool  and  add  I  to  2 
grams  of  the  finely  pulverized  sample.  Heat  gently  and  as  soon 
as  the  mass  softens,  stir  well  so  as  to  bring  the  whole  of  the  sample 
into  contact  with  the  alkali  before  it  is  strongly  heated,  then 
gradually  increase  the  heat,  stirring  frequently  to  keep  down  froth- 
ing, and  continue  heating  until  the  mass  becomes  colorless.  For 
samples  which  react  violently  when  stirred  into  the  alkali  as  above, 
a  smaller  proportion  of  nitrate  should  be  used  at  the  beginning,  or 
the  nitrate  may  be  omitted  at  the  start  and  added  after  the  sample 
has  been  mixed  with  the  fused  hydroxide.  If  the  substance  is  not 
quickly  oxidized  by  the  fusion  mixture,  small  amounts  of  pulverized 
nitrate  may  be  added  from  time  to  time  and  the  heating  continued 
with  frequent  stirring.  The  fusion  will  usually  be  perfectly  color- 
less when  the  oxidation  of  organic  matter  is  complete.  After  cool- 
ing, dissolve  the  fusion  in  water  in  a  beaker  or  casserole,  acidulate 
with  hydrochloric  acid  and  evaporate  to  dryness  to  expel  nitric 

*  Compt.  Rend.,  1892,  114,  318;    1899,  129,  1002.     See  also  Hempel :    Ber.  deut. 
chem.  Ges.,  1897,  30,  202,  and  Langbein  :  Ztschr.  angew.  Chem.,  1900,  1227,  1259. 
•\Ztschr.  angew.  Chem.,  1892,  393.     See  also  Graeffe  :  Ibid. ,  1904,  616. 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.         17 

and  nitrous  acids  and  heat  at  110°  to  dehydrate  silica  if  present. 
If  much  nitrate  or  silica  was  present  the  evaporation  with  hydro- 
chloric acid  should  be  repeated.  Dissolve  the  residue  in  cold  water, 
adding  a  few  drops  of  hydrochloric  acid,  filter  if  not  perfectly  clear, 
heat  the  solution  to  boiling  and  precipitate  at  the  boiling  tem- 
perature by  adding  a  solution  of  barium  chloride,  drop  by  drop 
with  constant  stirring,  until  in  considerable  excess.  Boil  5  or  10 
minutes  longer  and  allow  to  stand  in  a  hot  place  until  the  precipi- 
tate settles,  leaving  the  supernatant  liquid  perfectly  clear.  Filter 
and  wash  the  barium  sulphate,  first  with  water  acidulated  with 
hydrochloric  acid,  then  with  water  till  free  from  chlorides  and 
finally  ignite  and  weigh  with  the  usual  precautions. 

Notes  and  Precautions.  — The  reagents  should  be  tested  by  making 
a  "  blank  "  determination  with  sugar  or  cellulose.  Do  not  reject 
this  blank  if  no  precipitate  appears  when  the  barium  chloride  is 
added,  but  allow  the  solution  to  stand  over  night  to  ensure  the 
separation  of  any  small  amount  of  barium  sulphate  present,  then 
filter,  wash,  ignite  and  weigh,  and  subtract  the  weight  from  that  of 
the  precipitate  found  in  the  determination.  An  apparently  trifling 
amount  of  precipitate  in  the  "blank"  may  weigh  enough  to 
appreciably  affect  the  results.  If  the  fusion  is  heated  by  an  ordi- 
nary gas  flame  it  is  likely  to  absorb  sulphur  compounds  from  the 
latter.  To  avoid  this  use  an  alcohol  flame  for  heating  the  fusions. 
It  cannot  be  expected  that  the  blank  determination  should  show 
accurately  the  sulphur  absorbed  from  the  gas  flame.  Even  when 
an  alcohol  flame  is  used  and  the  sulphur  obtained  from  the  reagents 
is  deducted,  the  results  are  sometimes  too  high.  According  to 
Keiser  *  this  is  due  chiefly  to  the  fact  that  the  fused  alkali  takes 
up  some  silver  from  the  crucible  and,  on  acidifying  with  hydro- 
chloric acid,  the  silver  chloride  formed  is  held  in  solution  by  the 
excess  of  potassium  chloride  present  and  is  afterward  carried  down 
by  the  barium  sulphate,  increasing  the  weight  of  the  precipitate. 
Keiser  therefore  recommends  that  the  neutralized  solution  be 
cooled,  diluted  with  water  to  about  a  liter  and  allowed  to  stand. 
Any  silver  chloride  present  will  then  precipitate  and  may  be 
filtered  out  before  adding  barium  chloride. 

A  modification  which  is  often  useful  consists  in  heating  the  sub- 
stance with  strong  nitric  acid  until  a  considerable  part  of  the 
organic  matter  has  been  oxidized,  after  which  an  excess  of  alkali  is 

*  Amer.  Chem.  Journ. ,  1883,  5,  207. 


1  8  ORGANIC   ANALYSIS. 

added  and  the  mixture  transferred  to  a  crucible  and  carefully  fused 
until  oxidation  is  complete.  Hammarsten*  found  this  method  to 
give  the  same  results  as  the  methods  of  Liebig  and  of  Classon  when 
applied  to  casein,  egg  albumen  and  gelatin. 


OSBORNE'S  PEROXIDE 

Convert  about  10  grams  of  sodium  peroxide  into  hydroxide 
by  adding  to  it,  in  a  silver  or  nickel  crucible,  a  slight  excess  of 
water  and  heating  until  the  excess  is  boiled  off.  Allow  the  fusion 
to  cool  until  pasty,  then  add  one  to  two  grams  of  the  sample  and 
stir  it  into  the  alkali  as  quickly  as  possible.  Heat  gently  and  stir 
well  to  keep  down  the  frothing.  When  the  first  vigorous  action  is 
over,  heat  until  the  mass  fuses  and  stir  in  small  portions  of  fresh 
peroxide  until  decomposition  is  complete  and  the  fusion  is  practi- 
cally colorless.  Then  allow  to  cool,  dissolve  the  fusion  and  acidu- 
late with  hydrochloric  acid  ;  boil  to  destroy  any  excess  of  peroxide 
and  to  expel  chlorine  and  complete  the  determination  as  in  Liebig's 
method. 

Notes.  —  Blank  determinations  must  be  made  as  in  the  case  of 
the  Liebig  method,  and  the  same  precautions  observed  to  prevent 
the  absorption  of  sulphur  compounds  from  gas  flames  or  from  the 
air  of  the  laboratory.  There  is  also  the  same  danger  of  high  re- 
sults from  contamination  of  the  barium  sulphate  precipitate.  If 
silica  is  absent  the  acidulated  solution  need  not  be  evaporated  to 
dryness  since  no  nitrate  is  used  in  the  fusion.  For  all  such  cases 
the  method  is  considerably  more  rapid  than  that  of  Liebig,  though 
requiring  somewhat  more  careful  attention  during  the  fusion  of 
the  substance  with  the  alkaline  oxidizing  mixture.  This  method 
has  been  extensively  used  during  recent  years  in  the  analysis  of 
proteids,  food  materials  and  physiological  products  and  has  lately 
been  adopted  by  -the  Association  of  Official  Agricultural  Chemists. 
Several  methods  involving  the  use  of  sodium  peroxide  had  pre- 
viously been  proposed,  of  which  that  of  Asboth  J  is  perhaps  the 
best  known.  This  consists  in  mixing  the  substance  with  10  grams 
of  dry  sodium  carbonate  and  5  grams  of  peroxide  in  a  nickel  cruci- 
ble and  heating  carefully  at  first  and  then  more  strongly  until 
oxidation  is  complete.  After  dissolving  the  fusion  in  water  the 

*  Ztschr.  physiol.  Chem.,  1885,  9,  273. 

f  Journ.  Amer.  Chem.  Soc.,  1902,  24,  142. 

J  Chem.  Ztg.,  1895,  19,  2040. 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.          19 

solution  may  be  boiled  with  bromine  to  ensure  complete  oxida- 
tion. The  alkaline  mixture  here  used  being  much  less  fusible  than 
sodium  or  potassium  hydroxide,  the  method  should  be  used  only 
for  such  substances  as  do  not  readily  lose  sulphur  on  heating.  For 
such  substances  it  offers  the  advantage  of  simplicity  in  manipula- 
tion. 

BERTHELOT'S  OXYGEN  METHOD. 

Press  into  the  form  of  a  pellet  a  suitable  amount  of  the  substance 
(usually  one  to  three  grams),  introduce  into  the  bomb  calorimeter 
and  burn  in  oxygen  under  a  pressure  of  about  25  atmospheres  in 
the  same  manner  as  in  the  determination  of  heat  of  combustion.* 
Screw  into  the  exit  tube  of  the  bomb  a  coupling  carrying  a  delivery 
tube  of  about  0.5  mm.  internal  diameter  and  connect  with  a  wash- 
ing cylinder  containing  a  little  water.  Carefully  open  the  valve 
and  allow  the  gas  to  bubble  through  the  water  until  the  contents 
of  the  bomb  reach  atmospheric  pressure ;  then  disconnect,  open 
the  bomb  and  rinse  out  all  moisture  which  has  condensed  in  the 
chamber,  on  the  lining  of  the  cover,  or  on  the  rods  which  support 
the  combustion  capsule.  This  must  be  done  carefully  as  the 
rinsings  usually  contain  the  greater  part  of  the  sulphur.  In  order 
to  keep  down  the  volume  of  the  solution,  the  water  from  the  wash- 
ing cylinder  may  be  used  for  the  first  rinsing.  Dissolve  any  ash 
found  in  the  combustion  capsule  in  a  little  hydrochloric  acid,  re- 
move silica,  if  necessary,  and  add  the  solution  to  that  obtained  by 
rinsing  the  bomb.  Examine  the  lead  gasket  in  the  cover  of  the 
bomb  and  if  a  slight  film  of  sulphate  is  found,  wash  it  into  the 
main  solution.")"  Finally  boil  this  solution  down  to  the  desired 
volume,  neutralize  any  excessive  amount  of  hydrochloric  acid 
which  may  have  been  introduced  with  the  solution  of  the  ash,  filter 
if  necessary  and  determine  the  sulphuric  acid  by  precipitation  with 
barium  chloride  as  already  described. 

Notes.  —  This  method,  based  mainly  upon  the  suggestion  of  Ber- 
thelot  and  the  subsequent  work  of  Hempel,  has  been  applied  J  with 
very  satisfactory  results  to  a  number  of  animal  and  vegetable  sub- 
stances as  well  as  to  synthetic  organic  compounds  containing  sul- 

*  Atwater  and  Snell :  Jour.  Amer.  Chem.  Soc.,  1903,  25,  659. 

t  Such  minute  quantities  of  lead  sulphate  as  are  ordinarily  found  will  dissolve  readily 
in  the  solution  and  will  not  interfere  with  the  precipitation  of  sulphuric  acid  as  barium 
sulphate. 

\Jour.  Amer.  Chem.  Soc.,  1902,  24,  noo. 


20  ORGANIC   ANALYSIS. 

phur  in  forms  not  readily  oxidized  to  sulphuric  acid  —  e.  g.,  benzyl 
sulphide  and  diphenyl-thiourea.  So  far  as  tested,  no  cases  of  in- 
complete combustion  have  been  found.  If  this  were  suspected,  the 
gas  drawn  off  after  the  combustion  should  be  led  through  bromine 
water  or  an  alkaline  bromine  solution  and  the  washings  of  the 
bomb  added  to  this  solution  and  boiled  to  insure  oxidation  of  sul- 
phites to  sulphates.  Unless  the  bomb  is  emptied  gradually  in 
some  such  way  as  has  been  described,  there  is  danger  that  some 
sulphuric  acid  may  be  carried  away  mechanically  by  the  escaping 
gas.  This  is  probably  the  principal  cause  of  the  low  results  occa- 
sionally reported.  When  properly  carried  out  this  method  has 
several  important  advantages.  Combustion  takes  place  instanta- 
neously and  with  no  chance  for  the  escape  of  volatile  products. 
The  products  of  combustion  can  be  drawn  off  and  examined  as 
desired.  The  only  reagents  required  in  obtaining  the  sulphur  as 
sulphuric  acid  are  compressed  oxygen  and  hydrochloric  acid,  both 
of  which  are  easily  obtained  free  from  any  appreciable  amounts  of 
sulphur.  By  eliminating  the  use  of  alkali,  any  danger  of  absorp- 
tion of  sulphur  compounds  from  the  air  is  avoided,  and  no  con- 
siderable amount  of  foreign  salts  is  introduced  into  the  solution 
from  which  the  sulphuric  acid  is  to  be  precipitated. 

THE    DETERMINATION   OF    PHOSPHORUS. 

The  oxidation  of  organic  matter  for  the  determination  of  phos- 
phorus may  be  accomplished  by  any  of  the  methods  described  in 
connection  with  the  sulphur  determination.  It  is  of  course  un- 
necessary in  this  case  to  avoid  the  use  of  gas  flames  and  with  most 
substances  there  is  much  less  danger  of  loss  by  volatilization  than 
in  the  case  of  sulphur.  For  these  reasons  and  because  of  the  care 
required  in  dissolving  the  fused  residue  of  phosphates  left  in  the 
ignition  capsule,  the  compressed  oxygen  method  has  no  such 
marked  advantages  in  the  determination  of  phosphorus  as  in  that 

of  sulphur. 

ALKALI  METHODS. 

Liebig's  method  of  oxidation,  either  with  or  without  previous 
digestion  with  nitric  acid,  or  oxidation  by  peroxide  may  be  used 
but  the  use  of  sodium  carbonate,  as  in  the  following  method,  is 
generally  more  convenient. 

Mix  I  to  3  grams  substance  with  6  to  7  grains  dry  sodium  car- 
bonate in  a  platinum  dish  and  spread  3  or  4  grams  of  the  carbonate 


NITROGEN,  SULPHUR  AND  PHOSPHORUS.         21 

over  the  mixture.  Heat  over  a  Bunsen  burner,  carefully  at  first 
until  frothing  ceases,  then  strongly  until  the  mass  fuses.  To  the 
fusion  add  small  portions  of  pulverized  potassium  nitrate,  stirring 
thoroughly  with  a  platinum  rod  or  spatula,  until  the  mass  is  en- 
tirely colorless.  The  whole  amount  of  nitrate  required  does  not 
usually  exceed  one  gram.  After  cooling,  transfer  the  fusion  to  a 
beaker,  dissolve  in  water,  add  an  excess  of  nitric  acid  and  boil. 
Allow  the  solution  to  cool,  neutralize  with  ammonia,  add  a  few 
drops  of  nitric  acid  and  then  a  moderate  excess  of  molybdate 
solution.*  Digest  at  about  65°  for  an  hour  with  occasional  shak- 
ing or  stirring,  filter,  and  wash  with  cold  water  or  a  cold  solution 
of  ammonium  nitrate.  Test  the  filtrate  by  adding  more  molybdate 
solution  and  digesting  again  at  about  65°.  Place  the  beaker  used 
for  the  precipitation  under  the  funnel,  dissolve  the  precipitate 
through  the  paper  by  means  of  ammonia  and  wash  thoroughly 
with  water,  keeping  the  volume  of  the  solution  below  100  c.c. 
Nearly  neutralize  with  hydrochloric  acid,  cool  thoroughly  and  add 
a  moderate  excess  of  magnesia  mixture,f  drop  by  drop,  with  con- 
stant stirring.  After  15  minutes  add  10  c.c.  of  concentrated  am- 
monia or  its  equivalent ;  let  stand  four  to  twenty-four  hours  ;  filter, 
(preferably  on  an  asbestos  felt  in  a  Gooch  crucible)  wash  with 
dilute  ammonia  (2.5  to  5  Per  cent.)  until  practically  free  from 
chlorides,  ignite  and  weigh  as  magnesium  pyrophosphate. 

NEUMANN'S  ACID  METHOD.J 

To  I  to  3  grams  substance  in  a  Kjeldahl  flask  add  20  c.c.  con- 
centrated sulphuric  acid  and  10  grams  of  ammonium  nitrate  and 
heat  carefully  until  frothing  ceases;  or  heat  the  sample  first  with 
10  c.c.  fuming  nitric  acid,  then  cool  and  add  10  c.c.  of  sulphuric 
acid.  In  either  case  the  solution  is  boiled  with  successive  addi- 
tions wf  ammonium  nitrate  until  organic  matter  is  entirely  de- 
stroyed and  the  solution  is  colorless.  If  necessary,  as  many  grams 
of  ammonium  nitrate  may  be  added  as  the  number  of  c.c.  of  sul- 
phuric acid  used.  Cool  the  colorless  solution,  wash  it  out  into  a 

*Of  the  usual  molybdate  solution  containing  about  5  per  cent,  of  molybdic  acid, 
add  at  least  50  c.c.  for  every  decigram  of  phosphoric  acid  expected. 

flf  the  magnesia  mixture  is  of  the  usual  strength,  containing  about  5  per  cent,  of 
magnesium  chloride,  add  about  15  c.c.  for  each  decigram  of  phosphoric  acid  expected. 

J  DuBois  Reymond's  Archiv.  (physiol.  Abth.),  1897,  552  ;  1900,  159  ;  Abs.  Ghent. 
Centrbl.,  1900,  I.,  571.  See  also  Ztschr.  physiol.  Chetn.,  1900,  29,  146  ;  1902-3,  37 
115  ;  Journ.  Amer.  Chem.  Soc.t  1902,  24,  I  ico. 


22  ORGANIC   ANALYSIS. 

beaker,  neutralize  with  ammonia,  add  15  to  20  grams  of  ammon- 
ium nitrate  and  determine  phosphoric  acid  as  in  the  preceding 
method,  adding  a  somewhat  greater  excess  of  the  molybdate 
reagent. 

Notes.  —  This  method  has  been  found  to  give  good  results  with 
a  variety  of  foods  and  physiological  products.  The  large  amount 
of  sulphates  retards  the  formation  of  the  phosphomolybdate  pre- 
cipitate, but  by  using  liberal  quantities  of  ammonium  nitrate  and 
molybdate  reagent  and  allowing  at  least  an  hour  for  the  precipi- 
tation, the  whole  of  the  phosphoric  acid  is  obtained  without 
difficulty.  When  the  substance  is  first  treated  with  fuming  nitric 
acid,  a  smaller  quantity  of  sulphuric  acid  may  be  used  and  the 
phosphomolybdate  will  then  form  somewhat  more  quickly. 

In  applying  this  method  to  liquids  such  as  milk  or  urine,  a  suit- 
able amount  of  sample  (25  c.c.  milk  or  50  c.c.  urine,)  is  transferred 
to  the  flask,  20  c.c.  sulphuric  acid  added,  and  the  mixture  heated 
carefully  as  in  the  determination  of  nitrogen  until  the  sample  is  well 
charred  and  the  greater  part  of  the  water  has  been  boiled  off. 
Ammonium  nitrate  is  then  added  and  the  determination  is  com- 
pleted  as  above  described. 

For  additional  notes  on  this  method  and  a  discussion  of  methods 
for  the  separation  of  organic  and  inorganic  phosphorus  in  plant 
and  animal  products,  see  Hart  and  Andrews,  Amer.  Chem.  Journ. 
1903,  30,  470- 


CHAPTER  III. 

Alcohols. 

Alcohols  being  usually  defined  as  neutral  hydroxyl  derivatives 
capable  of  reacting  with  acids  to  form  esters,  it  follows  that  the 
most  characteristic  alcohol  reactions  are  those  involving  the  re- 
placement of  the  hydroxyl  by  an  acid  radical.  For  detailed  dis- 
cussions of  the  analytical  behavior  of  the  hydroxyl  radical  and  char- 
acteristic reactions  of  the  different  groups  of  alcohols,  the  reader  is 
referred  to  the  works  of  Hans  Meyer,*  while  Mulliken's  tables  | 
may  be  followed  in  the  systematic  identification  of  individual  pure 
preparations. 

Of  the  analytical  methods  involving  reactions  of  the  hydroxyl 
group,  the  more  important  are  those  in  which  the  acetyl  or  benzoyl 
ester  is  formed.  The  preparation  of  the  dinitrobenzoate  serves  for 
the  identification  of  ethyl  alcohol  as  described  below.  Quanti- 
tative methods  based  on  acetylation  will  be  described  in  the 
sections  on  glycerol  and  the  fatty  oils.  Often,  however,  esterifi- 
cation  is  either  not  quantitative  or  is  less  convenient  or  less  deli- 
cate than  other  methods  of  determination.  Thus  all  monatomic 
alcohols  containing  less  than  four  carbon  atoms  mix  freely  with 
water  and  are  not  readily  separated  from  it  but  may  be  deter- 
mined in  aqueous  solution  either  by  physical  methods  or  by  the 
behavior  of  the  acohols  on  oxidation. 

The  present  chapter  will  be  devoted  essentially  to  ethyl  alcohol 
and  glycerol.  The  processes  described  for  the  quantitative  deter- 
mination of  these  compounds  include  the  application  of  physical 
methods,  methods  of  oxidation  and  methods  of  acetylation.  In 
connection  with  the  determination  of  ethyl  alcohol,  special  methods 
for  the  detection  or  determination  of  a  few  of  its  more  important 
homologues  will  be  briefly  outlined.  For  more  extended  discus- 
sions of  the  analytical  chemistry  of  the  alcohols  the  work  of  Allen  J 
should  be  consulted. 

*  Analyse  und  Konstitutionsermittelung  organischer  Verbindungen,  Berlin,  1903. 
Determination  of  Radicles  in  Carbon  Compounds  (English  Edition  by  Tingle),  New 
York,  1903. 

f  Identification  of  Pure  Organic  Compounds,  New  York,  1 904. 

J  Commercial  Organic  Analysis,  Volume  I,  Philadelphia,  1898. 

23 


24  ORGANIC   ANALYSIS. 

ETHYL   ALCOHOL. 

Pure  ethyl  alcohol  is  a  colorless,  mobile  liquid  of  characteristic 
penetrating  odor  and  "hot"  pungent  taste,  boiling  at  about 
78.4°.  It  mixes  with  water  in  all  proportions  and  is  only  with 
great  difficulty  obtained  in  the  anhydrous  or  "absolute"  state. 
According  to  Allen  the  presence  of  as  small  a  proportion  as  0.5 
per  cent,  of  water  in  alcohol  is  indicated  by  the  pink  color  as- 
sumed by  the  liquid  on  introducing  a  crystal  of  potassium 
permanganate.  The  so-called  "  absolute  alcohol "  used  in  an- 
alytical operations  ordinarily  contains  from  0.2  to  I  per  cent  of 
water. 

Until  recently  the  density  of  anhydrous  alcohol  at  60°  F.  (15.55° 
C.)  was  considered  to  be  0.79381  referred  to  water  at  the  same 
temperature.  Squibb  obtained  alcohol  which,  while  not  absolutely 
anhydrous,  had  a  density  of  only  0.79350  at  60°  F.  indicating  that 
the  alcohol  previously  considered  absolute  must  have  contained  at 
least  O.I  per  cent,  of  water. 

On  mixing  alcohol  with  water  a  considerable  evolution  of  heat 
takes  place  and  the  volume  of  the  mixture  after  cooling  is  less 
than  the  sum  of  the  volumes  of  alcohol  and  water  mixed.  This 
contraction  is  not  uniformly  proportional  to  the  amount  of  alcohol 
in  the  mixture.  Hence  in  mixtures  of  water  and  alcohol  the  rela- 
tion between  the  percentages  of  alcohol  "  by  volume  "  and  "  by 
weight  "  varies  somewhat  with  the  strength  of  the  solutions.  These 
variations  together  with  the  differences  in  the  density  of  the  sup- 
posedly absolute  alcohol  used  as  standard  by  the  various  observers, 
account  for  the  small  discrepancies  found  on  comparing  the  com- 
monly used  tables  showing  the  relation  between  density  and  per- 
centage. Thus  the  table  of  Windisch  which  is  usually  given  in 
.German  text-books,  that  of  Allen  which  appears  to  be  based  on 
Hehner's  table,  and  that  of  Edgar  Richards  based  mainly  on  the 
work  of  Squibb,  is  each  different,  though  the  variations  are  rarely 
sufficient  to  be  noticeable  in  ordinary  work. 

Alcohol  dissolves  many  organic  substances  which  are  not  soluble 
in  water,  but  inorganic  compounds  insoluble  in  water  are  usually 
also  insoluble  in  alcohol.  As  a  rule  chlorides,  bromides,  iodides, 
and  acetates  are  soluble  in  fairly  strong  alcohol,  while  carbonates, 
borates,  sulphates,  phosphates,  oxalates  and  tartrates  are  only  very 
sparingly  soluble. 


ALCOHOLS.  25 

DETECTION  AND  IDENTIFICATION. 
Liebens  Iodoform  Test. 

The  "  iodoform  test"  while  not  distinctive  is  often  useful.  It 
may  be  carried  out  as  follows:  To  10  c.c.  of  the  clear  liquid  to  be 
tested,  add  5  or  6  drops  of  10  per  cent,  solution  of  sodium  or  pot- 
assium hydroxide,  heat  to  about  50°  C.  and  add  drop  by  drop  with 
constant  shaking  a  saturated  solution  of  iodine  in  aqueous  potas- 
sium iodide  until  the  liquid  becomes  just  permanently  yellowish- 
brown,  then  carefully  decolorize  by  adding  more  of  the  hydroxide 
solution,  avoiding  excess.  If  alcohol  were  present,  iodoform  grad- 
ually separates  out  as  a  yellow  or  yellowish-white  crystalline  de- 
posit. Even  when  very  little  iodoform  is  produced  its  odor  can 
usually  be  recognized.  While  the  test  is  quite  delicate,  the  appear- 
ance of  a  precipitate  of  iodoform  does  not  prove  the  presence  of 
ethyl  alcohol  since  it  may  result  from  various  other  compounds, 
especially  acetone,  aldehydes  and  the  propyl  and  butyl  alcohols. 
If  the  original  liquid  contained  carbohydrate  or  organic  acid  it 
should  be  neutralized,  distilled  and  the  first  portion  of  the  distillate 
used  for  the  test. 

Ethyl  Dinitrobenzoate  Test. 

As  a  specific  test  for  ethyl  alcohol  Mulliken  *  recommends  the 
preparation  of  Ethyl  3,  5-Dinitrobenzoate  as  follows  : 

Heat  together  gently  in  a  three-inch  test-tube  held  over  a  small 
flame,  0.15  gm.  of  3,  5-dinitrobenzoic  acid  and  0.20  gm.  of  phos- 
phorus pentachloride.  When  signs  of  chemical  action  are  seen, 
remove  the  heat  for  a  few  seconds.  Then  heat  again,  boiling  the 
liquefied  mixture  very  gently  for  one  minute.  Pour  out  on  a  very 
small  watchglass  and  allow  to  solidify.  As  soon  as  solidification 
occurs,  remove  the  liquid  phosphorus  oxychloride,  with  which  the 
crystalline  mass  is  impregnated,  by  rubbing  the  latter  between  two 
small  pieces  of  porous  tile.  Place  the  powder  in  a  dry  five  or  six- 
inch  test-tube.  Allow  four  drops  of  the  alcohol  (which  must  con- 
tain not  more  than  about  10  per  cent,  of  water)  to  fall  upon  it,  and 
then  stopper  the  tube  tightly  without  delay.  Immerse  the  lower 
part  of  the  test-tube  in  water  having  a  temperature  of  75°-85°. 
Shake  gently,  and  continue  the  heating  for  ten  minutes. 

To  purify  the  ester  produced  in  the  reaction,  crush  with  a  stir- 
ring rod  any  hard  lumps  which  may  form  when  the  mixture  cools 

*  Loc.  cit.,  p.  1 68. 


26  ORGANIC   ANALYSIS. 

and  boil  gently  with  15  c.c.  of  methyl  alcohol  (2:1)  until  all  is  dis- 
solved or  for  a  minute  or  two.  Filter  boiling  hot  if  the  solution  is 
not  clear.  Cool,  shake  and  filter.  Wash  with  3  c.c.  cold  methyl 
alcohol  (2:1).  Recrystallize  from  9  c.c.  of  boiling  methyl  alcohol 
(2:1).  Wash  with  2  c.c.  of  the  same  solvent.  Spread  out  the 
product  on  a  piece  of  tile.  Allow  to  become  air-dry,  and  determine 
the  melting  point. 

Ethyl  3,  5-Dinitrobenzoate,  the  product  of  this  test  crystallizes 
in  white  needles  melting  at  92°-93°  (uncorr.). 

The  corresponding  derivatives  of  methyl,  propyl,  butyl,  and 
isobutyl  alcohols  melt  at  107.5°  (uncorr.),  730-73-50  (uncorr.),  64° 
(uncorr.)  and  83°-83.5°  (uncorr.)  respectively. 

DETERMINATION  BY  THE  SPECIFIC  GRAVITY  METHOD. 

This  method  which  is  the  most  generally  applicable  and  the 
most  accurate  for  the  determination  of  alcohol  in  ordinary  alcoholic 
liquors,  consists  in  obtaining  the  alcohol  mixed  only  with  water 
and  determining  the  amount  of  alcohol  present  by  finding  the 
specific  gravity  of  this  aqueous  solution.  It  will  be  convenient  to 
describe  first  the  method  as  applied  to  fermented  liquors  and  then 
note  the  modifications  to  be  introduced  in  the  analysis  of  other 
alcohol  preparations. 

Alcohol  in  Fermented  Liquors. 

Dry  and  weigh  a  100  c.c.  graduated  flask.  Fill  to  the  holding 
mark  with  the  sample*  and  weigh.  Transfer  to  a  distilling 
flask  of  250  to  300  c.c.  capacity,  rinsing  out  the  measuring  flask 
with  about  50  c.c.  of  water.  Add  about  one-tenth  gram  of  cal- 
cium carbonate  to  neutralize  any  acid  present,  or  carefully  neu- 
tralize by  means  of  dilute  sodium  or  potassium  hydroxide,  connect 
with  a  well  cooled  condenser  and  distil,  collecting  the  distillate  in 
the  flask  used  for  measuring  the  sample.  When  the  receiver  is 
filled  to  the  mark  remove  it,  wipe  the  outside  with  a  dry  cloth  and 
weigh.  By  means  of  a  pyknometer  determine  accurately  (to  five 
decimal  places)  the  specific  gravity  of  the  distillate  at  15.55°  C. 
(60°  F.)  compared  with  recently  boiled  distilled  water  at  the  same 

*  If  the  sample  contains  much  carbon  dioxide  this  should  be  removed  as  far  as 
possible  in  advance  by  shaking  the  liquor  in  a  large  flask  at  room  temperature. 


ALCOHOLS. 


27 


PERCENTAGE  OF  ALCOHOL  BY  WEIGHT  AND  BY  VOLUME. 

[Recalculated  from  the  determinations  of   Gilpin,  Drink  water  and  Squibb,  by 

EDGAR  RICHARDS.] 


>, 

I* 

% 

o  a 
O) 

Per  Cent.  Alcohol 
by  Volume. 

Per  Cent.  Alcohol 
by  Weight. 

Specific  Gravity 
at  §8°  F. 

PerCent.  Alcohol 
by  Volume. 

PerCent  Alcohol 
by  Weight. 

Specific  Gravity 
at  |8°  F. 

Per  Cent.  Alcohol 
by  Volume. 

Per  Cent.  Alcohol 
by  Weight. 

I.OOOOO    O.OO 

o.co 

0.99281 

5-00 

4.00 

0.98660 

10.00 

8.04 

0.99984    .10 

.08 

268 

.10 

.08 

649 

.10 

.12 

968     .20 

.16 

255 

.20 

.16 

637 

.20 

.20 

953 

•30 

.24 

241 

.30 

.24 

626 

•30 

.29 

937 

.40 

•32 

228 

.40 

•32 

614 

.40 

•37 

.99923 

0.50 

0.40 

.99215 

5-5°     4.40 

.98603   10.50 

8.45 

907 

.60 

.48 

202 

.60 

.48        592     .60 

•53 

892 

.70 

.56 

189 

.70 

•56 

580 

.70 

.61 

877 

.80 

.64 

175 

.80 

.64 

569 

.80 

.70 

861 

.90 

•71 

162 

.90 

.72 

557 

.90 

•78 

.99849 

1.  00 

0.79 

.99H9 

6.00 

4.80 

.98546 

11.00 

8.86 

834 

.10 

.87 

136 

.10 

.88 

535 

.10 

.94 

819 

.20 

•95      123 

.20 

.96 

524 

.20 

9.02 

805 

•30 

1.03      in 

•30 

5-05 

5i3 

.30 

.11 

790 

.40 

.  1  1      098 

.40 

•13 

502 

.40 

.19 

•99775 

1-50 

1.19 

.99085 

6.50 

5-21 

.98491 

11.50 

9.27 

760 

.60 

•27 

072 

.60 

.29 

479 

.60 

•35 

745 

.70 

•35 

059 

.70 

•37 

468 

.70 

•43 

73i 

.80 

•43 

047 

.80 

•45 

457 

.80 

•51 

716 

.90 

•51 

034 

.90 

•53 

446 

.90 

•  59 

.99701 

2.00 

i-59 

.99021 

7.00 

5.61 

.98435 

12.00 

9.67 

687 

.10 

.67 

009 

.10 

.69 

424 

.10 

•75 

672 

.20 

•75 

.98996 

.20 

•77 

413 

.20 

•83 

658 

•30 

.83 

984 

•3° 

.86 

402 

•30 

.92 

643 

.40 

.91 

971 

.40 

.94 

39i 

.40 

10.00 

0.99629 

2.50 

1.99 

.98959 

7-50 

6.02 

.98381 

12.50 

10.08 

615    .60 

2.07 

947 

.60 

.10 

370 

.60 

.16 

600 

.70 

.15 

934 

•  70 

.18 

359 

.70 

.24 

586 

.80 

•23 

922 

.80 

.26 

348 

.80 

•33 

57i 

.90 

•31 

909 

.90 

•34 

337 

.90 

.41 

•99557 

3.00 

2.39 

.98897 

8.00 

6.42 

.98326 

13.00 

10.49 

543 

.10 

•47 

885 

.10 

.50 

3i5 

.10 

•57 

529 

.20 

•55 

873 

.20 

.58 

305 

.20 

.65 

515 

•30 

•64 

861 

.30 

.67 

294 

•30 

•74 

5oi 

.40 

.72 

849 

.40 

•75 

283 

.40  i    .82 

.99487 

3-50 

2.80 

.98837 

8.50 

6.83 

•98273 

13.50 

10.90 

473 

.60 

.88 

825 

.60 

.91 

262 

.60 

.98 

459 

.70 

.96 

813 

.70 

•99 

251 

•  70 

1  1.  06 

445 

.80 

3-04 

801 

.80 

7.07 

240 

.80 

•15 

431 

.90 

.12 

789 

.90 

•15 

230 

.90 

•23 

.99417 

4.00 

3-2b 

.98777 

9.00 

7-23 

.98219 

14.00 

11.31 

403 

.10 

.28 

765 

.10 

•3i 

209 

.10 

•39 

390 

.20 

.36 

754 

.20 

•39 

198 

.20 

•47 

376 

•30 

.44 

742 

•30 

•48 

188 

•30 

.56 

363 

.40 

•52 

730 

.40 

•56 

177 

.40 

.64 

•99349 

4.50 

3.60 

.98719 

9.50 

7.64 

.98167 

14.50 

11.72 

335 

.60 

.68 

707 

.60 

.72 

156 

.60 

.80 

322 

.70 

.76 

695 

.70 

.80 

146 

.70 

.88 

308 

.80 

.84 

683 

.80 

.88 

135 

.80 

.97 

295 

.90 

.92 

672 

.90 

.96 

125 

.90 

12.05 

28 


ORGANIC   ANALYSIS. 


PERCENTAGE  OF  ALCOHOL  BY  WEIGHT  AND  BY  VOLUME.  —  Contimied. 

[Recalculated  from  the  determinations  of  Gilpin,  Drinkwater  and  Squibb,  by 

EDGAR  RICHARDS.] 


>> 

L 

0. 

y  "ro 

*3  4J 

Y 

& 

OT 

Per  Cent.  Alcohol 
by  Volume. 

Per  Cent.  Alcohol 
by  Weight. 

Specific  Gravity 
at  §g°  F. 

Per  Cent.  Alcohol 
by  Volume. 

Per  Cent.  Alcohol 
by  Weight. 

>, 

8fe 

00   . 
«§? 
|- 
W 

Per  Cent.  Alcohol 
by  Volume. 

Per  Cent.  Alcohol  j 
by  Weight. 

0.98114 

15.00 

12.13 

0.97608 

2O.OO 

16.26 

0.97097 

25.00 

20.43 

IO4 

.10 

.21 

598 

.10 

•34 

086 

.10 

•51 

093 

.20 

.29 

588 

.20 

.42 

076 

.20 

.60 

083 

•30 

.38 

578 

•30 

.51 

065 

•30 

.68 

073 

.40 

.46 

568 

.40 

•  59 

055 

.40 

•  77 

.98063 

I5-50 

12.54 

.97558 

20.50 

16.67 

.97044 

25.50     20.85 

052 

.60 

.62 

547 

.60 

.75 

033 

.60 

•93 

042 

.70 

•70     537 

.70 

.84 

023 

•70 

21.02 

032 

.80 

•79 

527 

.80 

.92 

012 

.80 

.10 

021 

.90 

.87 

517 

.90 

17.01 

OOI 

.90 

.19 

.980II 

16.00 

12.95   .97507 

21.  OO 

17.09 

.96991 

26.OO     21.27 

OOI 

.10 

I3-03     497 

.IO 

•17 

980 

•i°     -35 

.97991 

.20 

.12     487 

.20 

.26 

969 

.20     .44 

980 

•30 

.20     477 

•30 

•34 

959    -3°     .52 

970 

.40 

.29     467 

.40 

•43 

949  !   .40     .61 

.97960   16.50 

13-37   -97457 

21.50 

17-51 

.96937   26.50  !  21.69 

950    .60 

•45 

446 

.60 

•59 

926 

.60     .77 

940   .70 

•53 

436 

.70 

.67 

915 

.70     .86 

929 

.80 

.62 

426 

.80 

•  76 

905 

.80     .94 

919 

.90 

.70     416 

.90 

•84 

894 

.90   22.03 

.97909  17.00 

13.78  ||  .97406 

22.OO 

17.92 

.96883 

27.00     22.11 

899    .10 

.86  !    396 

.IO 

18.00 

872 

.10       .20 

889 

.20 

•94 

386 

.20 

.09 

861 

.20       .28 

879 

•  3° 

14.03 

375 

•3° 

.17 

850 

•30       .37 

869    .  40 

•ii     365 

.40 

.26 

839 

•40       -45 

.97859  17.50 

14.19 

•97355 

22.50 

18.34 

.96828 

27.50     22.54 

848    .60    .27 

345 

.60 

.42 

816 

.60       .62 

838   .70  1  .35 

335 

.70 

•51 

805 

•70       .71 

828   .80    .44 

324 

.80 

•59 

794    -80     .79 

818    .90 

•52 

3H 

.90 

.68 

783 

.90     .88 

.97808   18.00 

14.60 

.97304 

23.00 

18.76 

.96772 

28.00   22.96 

798    .10 

.68 

294 

.10 

.84 

761 

.10     .04 

788 

.20 

•77 

283 

.20 

.92 

749 

•  20     .13 

778 

•3° 

.85 

273 

•3° 

19.01 

738 

.30       .21 

768 

.40 

.94 

263 

.40 

.09 

726 

.40 

•30 

.97758 

18.50 

15.02 

.97253 

23.5° 

19.17 

.96715 

28.50 

23.38 

748 

.60 

.10 

242 

.60 

•25 

704 

.60 

•47 

738 

.70 

.18 

232 

.70 

•34 

692 

.70 

•55 

728 

.80 

.27       222 

.80 

.42 

68  1 

.80 

.64 

718 

.90 

.38 

211 

.90 

•51 

669 

.90 

.72 

.97708 

19.00 

15-43 

.97201 

24.00 

19-59 

.96658 

29.00   23.81 

698 

.10 

•51 

191 

.10 

.67 

646 

.10     .89 

688 

.20 

-59 

1  80 

.20 

.76 

635 

.20       .98 

678 

•3° 

.68 

170 

•3° 

.84 

623 

.30     24.06 

668 

.40 

.76  1   159 

.40 

•93 

611 

.40       .15 

.97658 

19.50 

15.84 

.97149 

24.50 

20.01 

.96600 

29.50     24.23 

648 

.60 

•93 

139 

.60 

.09 

587 

.60       .32 

638 

.70 

16.01      128 

.70 

.18 

576 

.70       .40 

628 

.80 

.09 

118 

.80 

.26 

564 

.80 

•49 

618 

.90 

.18 

107 

.90 

•35 

553 

.90       -57 

ALCOHOLS.  29 

temperature.  Find  in  the  accompanying  table  *  the  percentages  of 
alcohol  by  weight  and  by  volume  corresponding  to  this  specific 
gravity. 

The  percentage  by  weight  of  alcohol  in  the  distillate,  multiplied 
by  the  weight  of  the  latter  shows  the  actual  weight  of  alcohol  dis- 
tilled over  and  this  divided  by  the  weight  of  sample  taken,  gives 
the  percentage  by  weight  of  alcohol  in  the  liquor.  The  percentage 
by  volume  is  of  course  the  same  for  the  original  sample  as  for 
the  distillate,  if  both  are  measured  in  the  same  flask  at  the  same 
temperature. 

Alcohol  in  Other  Solutions. 

Distilled  Liquors.  —  Take  a  smaller  amount  of  the  sample  so  as 
to  have  not  over  25  grams  of  alcohol  present.  There  should  be  no 
acid  present  to  require  neutralization.  Dilute  the  portion  taken  for 
analysis  to  150  c.c.  in  the  distilling  flask,  distil  until  100  c.c.  have 
passed  over  and  complete  the  determination  as  described. 

Cordials,  bitters,  etc.,  which  are  likely  to  contain  high  percentages 
of  both  alcohol  and  extractive  matters,  should  be  diluted  if  neces- 
sary and  treated  as  described  for  fermented  liquors. 

Preparations  containing  chloroform,  ether,  or  essential  oils,  may  be 
treated  as  follows  :f  Dilute  25  c.c.  of  the  sample  with  water  to 
about  100  c.c.  in  a  separatory  funnel,  add  sodium  chloride  to  satu- 
ration and  then  50  to  80  c.c.  of  light  petroleum  distillate  (boiling 
below  60°).  Shake  vigorously  for  5  minutes,  allow  to  stand  for  half 
an  hour,  draw  off  the  lower  layer  into  another  separatory  funnel, 
wash  again  in  the  same  way  with  a  small  amount  of  the  petroleum 
ether  and  then  draw  off  into  a  distillation  flask.  Unite  the  petro- 
leum ether  layers,  wash  with  two  successive  portions  each  25  c.c. 
of  water  saturated  with  salt,  adding  these  washings  to  the  main 
solution.  Neutralize,  if  necessary,  and  distil  in  the  usual  way. 

Notes  on  the  Specific  Gravity  Method. 

When  the  distillation  is  carried  out  as  above  described,  100  c.c. 
being  distilled  from  an  original  volume  of  about  150  c.c.  containing 
not  more  than  25  grams  of  alcohol,  the  distillation  may  be  carried 
on  at  a  fairly  rapid  rate.  There  is  no  danger  that  the  whole  of 
the  alcohol  will  not  be  distilled  over,  and  if  the  condenser  is  kept 

*  Condensed  from  the  tables  adopted  by  the  Association  of  Official  Agricultural 
Chemists,  Bulletins  46  and  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 
|  Thorpe  and  Holmes  :  Journ.  Chem.  Soc.,  1903,  83,  314. 


30  ORGANIC   ANALYSIS. 

cooled  to  room  temperature  and  is  properly  connected  with  the 
receiver,  only  traces  of  alcohol  need  be  lost  by  evaporation.  The 
delivery  tube  of  the  condenser  may  either  extend  well  into  the 
neck  of  the  receiving  flask  or  may  be  connected  with  it  by  means 
of  an  "  adapter."  If  unusual  precautions  to  avoid  evaporation  are 
desired  the  receiver  may  be  closed  with  a  stopper  having  one  hole 
to  admit  the  adapter  and  another  fitted  with  a  small  reflux  con- 
denser, which  can  be  rinsed  with  a  few  drops  of  water  toward  the 
end  of  the  operation. 

It  is  necessary  to  exercise  great  care  in  the  determination  of  the 
specific  gravity  of  the  distillate,  especially  as  regards  the  tempera- 
ture at  which  the  pyknometer  is  filled.  If  this  is  different  from  the 
temperature  for  which  the  table  is  constructed,  each  weighing  must 
be  separately  reduced  to  standard  temperature  since  alcohol  and 
water  do  not  expand  at  the  same  rate.  The  safest  way,  both  in 
calibrating  the  pyknometer  and  in  making  the  determination,  is  to 
cool  the  liquid  to  a  little  below  1 5. 5°,  fill,  and  allow  the  temperature 
to  rise  slowly  until  the  proper  point  is  reached,  then  adjust  the 
pyknometer,  wipe  it  dry  on  the  outside  and  weigh.  Never  hold  the 
pyknometer  in  such  a  manner  that  the  temperature  of  its  contents 
can  be  affected  by  the  warmth  of  the  hand.  Find  the  weight  of 
water  which  the  pyknometer  holds  at  15.5°*  and  use  this  as 
divisor  in  estimating  the  specific  gravity  of  the  distillate.  Empty 
and  dry  the  pyknometer,  fill  with  distillate  at  15.5°  and  weigh. 
If  both  observations  are  made  at  15.5°,  the  weight  of  distillate 
divided  by  that  of  water  gives  the  desired  specific  gravity.  If  the 
pyknometer  is  filled  with  distillate  at  a  few  degrees  above  or  be- 
low 15.5°,  the  weight  must  be  divided  by  the  weight  of  water  at 
15.5°  to  find  the  "  apparent  specific  gravity  ",  then  correct  this  by 
the  following  empirical  formula  given  by  Allenf  in  which  D  is  the 
specific  gravity  at  i$.5°,D'  the  "  apparent"  specific  gravity,  and  d 
the  difference  (in  degrees  centigrade)  between  15 -5°  an<^  the  tem- 
perature at  which  D'  was  determined  : 


D= 


Y  i  -  D'  \ 

(  0.00014  +  —  ). 

V  150    ) 


*  If  for  any  reason  the  water  is  slightly  above  or  below  15.5°,  note  the  temperature 
accurately,  divide  the  weight  of  water  found  by  the  weight  of  I  c.c.  at  that  tempera- 
ture and  multiply  by  0.999055.  The  following  may  be  taken  as  the  weight  of  I  c.c. 
of  water  in  grams  at  the  temperatures  given:  13°,  0.999410  ;  14°,  0.999278;  15°, 
0-999I34;  16°,  0.998976;  17°,  0.998808;  18°,  0.998629;  19°,  0.998438;  20°, 
0.998235  ;  21°,  0.998023;  22°,  0.997801  ;  23°,  0.997568. 

f  Commercial  Organic  Analysis,  Vol.  I.  (3d  Ed.),  p.  93. 


ALCOHOLS.  31 

The  correction  is  added  to,  or  subtracted  from,  the  "  apparent 
specific  gravity"  according  as  the  pyknometer  was  filled  at  a  tem- 
perature above  or  below  the  standard. 

The  specific  gravity  of  the  distillate  being  known  to  five  decimal 
places,  its  alcohol  content  by  weight  and  by  volume  can  be  found 
to  the  second  decimal  place  by  intercalculation  between  the  values 
given  in  the  table. 

Pyknometers.  —  In  using  the  ordinary  form  of  specific  gravity 
bottle  having  a  simple  ground  glass  stopper  with  capillary  bore, 
care  must  be  taken  that  no  minute  bubbles  of  air  are  entrapped 
when  the  stopper  is  inserted.  This  may  be  largely  avoided  by 
grinding  the  lower  end  of  the  stopper  either  convex  or  concave. 
When  a  pyknometer  of  this  form  is  allowed  to  stand  at  a  higher 
temperature  than  that  at  which  it  has  been  filled,  the  expansion 
forces  some  of  the  liquid  out  upon  the  top  of  the  stopper,  whence 
it  may  be  lost  either  mechanically  or  by  evaporation.  Since  the 
temperature  of  the  laboratory  is  usually  considerably  above  15.5°, 
the  weighing  should  be  made  as  promptly  as  possible.  The 
Geissler  pyknometer  has  a  thermometer  inserted  in  the  ground 
glass  stopper  so  that  the  temperature  of  the  contents  can  be  read 
directly  at  any  time.  A  side  tubulure  of  small  bore  provides  for 
the  overflow  at  constant  volume  and  is  fitted  with  a  ground  glass 
cap,  so  that  when  the  adjustment  has  been  made  there  is  no  danger 
of  a  loss  of  contents  in  case  the  pyknometer  is  allowed  to  stand 
for  a  time  at  a  higher  temperature.  Squibbs'  pyknometer  *  has  a 
body  like  an  ordinary  specific  gravity  bottle,  but  the  ground  neck 
is  fitted  with  a  narrow  graduated  stem  instead  of  a  stopper.  Ad- 
justment is  secured  by  removing  small  portions  of  liquid  from  this 
stem  by  means  of  a  capillary  pipette  and  narrow  strips  of  blotting 
board.  The  construction  of  the  instrument  makes  it  easy  to  ob- 
serve when  the  contents  are  at  the  same  temperature  as  the  sur- 
rounding medium,  since  as  long  as  any  expansion  or  contraction 
is  taking  place,  the  column  of  liquid  will  move  up  or  down  in  the 
stem.  This  instrument,  like  that  of  Geissler,  avoids  all  inconve- 
nience from  overflow  during  weighing.  It  is  especially  useful 
when  one  wishes  to  use  the  same  pyknometer  for  determinations 
of  specific  gravity  at  different  temperatures.  Ostwald  objects  to 
any  pyknometer  having  a  tight  stopper  on  the  ground  that  "  as 
the  conical  angle  of  the  stopper  must  be  small  in  order  to  get  it  to 

* Journ.  Amer.  Ghent.  Soc.,  1897,  19,  ill. 


32  ORGANIC   ANALYSIS. 

fit  tight,  a  slight  elastic  deformation  of  the  neck  causes  a  consid- 
erable displacement  of  the  stopper  in  the  direction  of  its  axis,"  thus 
introducing  a  source  of  error  not  easily  avoided.  He  therefore 
recommends*  a  modification  of  the  Spengel  pyknometer  which  has 
now  come  into  quite  common  use.  With  care  in  avoiding  the 
sources  of  error  which  have  been  mentioned,  any  standard  form  of 
pyknometer  should  give  satisfactory  results. 

DETERMINATION  BY  THE  BOILING  POINT  METHOD. 

In  mixtures  of  alcohol  and  water  containing  no  appreciable 
amount  of  other  volatile  substances  and  only  small  quantities  of 
dissolved  solids,  the  difference  between  the  boiling  point  of  the 
mixture  and  that  of  pure  water  under  the  same  conditions,  gives  a 
measure  of  the  percentage  of  alcohol  present.  For  the  rapid  de- 
termination of  alcohol  on  this  principle,  several  forms  of  ebullio- 
scope  have  been  devised.  The  liquid  to  be  tested  is  boiled  under 
a  reflux  condenser  while  a  thermometer  bulb  is  fixed  just  above 
the  surface  of  the  liquid  so  as  to  be  entirely  surrounded  by  the 
vapor.  The  more  common  technical  forms,  such  as  those  of  Pohl 
and  Kappeller,  have  scales  reading  percentage  of  alcohol  instead 
of  thermometer  scales.  Water  is  first  boiled  in  the  apparatus  and 
the  scale  adjusted  so  that  the  mercury  stands  at  zero.  If  then  the 
water  be  removed  and  the  sample  introduced  and  brought  to  boil- 
ing under  the  same  barometric  conditions,  the  point  reached  by  the 
mercury  column  shows  the  amount  of  alcohol  present. 

Wileyf  uses  a  delicate  differential  thermometer  with  an  appa- 
ratus similar  to  that  employed  for  the  determination  of  molecular 
weights  by  the  boiling  point  method.  Up  to  five  per  cent,  of 
alcohol,  the  depression  of  the  boiling  point  is  said  to  be  so  regular 
that  the  results  are  entirely  satisfactory  for  practical  work.  In  the 
ebullioscopes  bearing  scales  graduated  in  terms  of  alcohol,  the 
variations  in  the  boiling  point  curve  at  the  higher  percentages  are, 
of  course,  allowed  for.  The  boiling  point  method  is  very  rapid 
and  gives  results  sufficiently  accurate  for  many  purposes.  Refer- 
ence may  be  made  to  Vaubel  J  for  a  general  discussion  of 
methods  based  on  the  determination  of  the  boiling  point  and  to 

*Ostwald:  Physico-Chemical  Measurements  (translated  by  Walker),  New  York, 
1894.  The  Sprengel-Ostwald  pyknometer  is  also  described  by  Getman  :  Laboratory 
Exercises  in  Physical  Chemistry,  New  York,  1904. 

•\Journ.  Amer.  Chem.  Soc.,  1896,  18,  1063. 

J  Qaantitative  Bestimmung  organischer  Verbindungen,  Berlin,  1902. 


ALCOHOLS.  33 

Freyer  *   for  experimental  results  on  the   influence   of  dissolved 
solids  in  the  ebullioscopic  determination  of  alcohol. 

DETERMINATION  BY  OXIDATION. 

Under  suitable  conditions  ethyl  alcohol  can  be  quantitatively 
oxidized  to  acetic  acid  by  means  of  potassium  dichromate  in  the 
presence  of  sulphuric  acid.  The  amount  of  alcohol  can  then  be 
ascertained  either  by  determining  the  amount  of  dichromate  re- 
duced f  or  by  distilling  and  titrating  the  acetic  acid  formed. J 
The  conditions  of  oxidation  must  be  carefully  regulated  and  as  a 
rule  the  method  is  used  only  for  the  determination  of  very  small 
amounts  of  alcohol,  the  specific  gravity  method  being  preferable 
for  the  examination  of  any  but  very  dilute  solutions.  The  alcohol 
must  of  course  be  separated  by  distillation  from  any  other  oxidiz- 
able  matter  before  the  oxidation  method  can  be  applied.  A 
comparison  of  the  results  obtained  by  oxidation  with  those  shown 
by  the  specific  gravity  method  may  be  useful  in  demonstrating  the 
presence  of  homologous  alcohols. 

DETECTION  AND  DETERMINATION  OF  HOMOLOGOUS  ALCOHOLS. 

Methyl  Alcohol. 

In  many  countries, "  methylated  spirit "  (usually  a  mixture  of  9 
parts  ethyl  alcohol  with  I  part  commercial  wood  spirit)  is  exempt 
from  the  taxes  levied  upon  other  forms  of  alcohol.  The  former  is 
therefore  much  cheaper  and  there  is  great  inducement  to  substi- 
tute it  for  pure  ethyl  alcohol  in  the  preparation  of  tinctures,  etc., 
where  its  use  is,  however,  highly  objectionable  on  account  of  the 
toxicity  of  the  methyl  alcohol.  Under  such  circumstances  the  de- 
tection of  small  amounts  of  methyl  alcohol  or  wood  spirit  is  often 
of  importance.  In  the  United  States,  "methylated  spirit"  is  not 
exempt  from  taxation  ^1904)  and  delicate  tests  for  its  detection  are 
therefore  of  much  less  practical  importance.  Methyl  alcohol  itself 
may  be  fraudulently  substituted  for  ethyl  alcohol  but  this  would 
be  profitable  only  when  considerable  quantities  were  involved  and 
in  such  cases  the  presence  of  the  methyl  alcohol  should  be  shown 
by  quantitative  methods. 

*  Ztschr.  angew.  Chem.,  1896,  654. 

f  Hehner  :  Analyst,  1887,  12,  25.  Benedict  and  Norris  :  Journ.  Amer.  Chem. 
Soc.,  1898,  20,  293. 

|  Dupre  :  Journ.  Chem.  Soc.,   1867,  20,  495. 


34  ORGANIC   ANALYSIS. 

Aqueous  solutions  of  methyl  alcohol  have  specific  gravities  so 
close  to  those  of  ethyl  alcohol  of  the  same  strength  that  the  total 
amount  of  the  two  alcohols  can  be  determined  by  distilling,  taking 
the  specific  gravity  of  the  distillate  and  calculating  the  percentage 
from  the  ethyl  alcohol  table.  This  distillate  may  then  be  exam- 
ined for  methyl  alcohol  by  one  of  the  following  methods. 

Color  Reactions. — The  color  reactions  now  commonly  used  * 
depend  upon  oxidation  of  the  methyl  alcoholto  formaldehyde  which 
is  then  detected  by  one  of  the  tests  described  in  the  next  chapter. 
These  tests  can  be  depended  upon  only  when  very  carefully  car- 
ried out  with  attention  to  the  precautions  given  by  Mulliken  and 
Scudder,  |  while  great  care  must  also  be  exercised  in  the  interpre- 
tation of  the  tests  inasmuch  as  positive  reactions  for  methyl  alco- 
hol have  been  obtained  %  in  the  case  of  a  number  of  genuine 
wines  and  brandies. 

The  Iodide  Method  of  'Lam  §  is  based  upon  the  fact  that  methyl 
iodide  is  considerably  heavier  than  ethyl  iodide,  the  former  having 
at  15°  a  specific  gravity  of  2. 2677,  while  that  of  the  latter  is  1-9444. 
The  mixture  of  alcohols  to  be  tested  is  treated  with  red  phosphorus 
and  iodine  and  the  resulting  iodides  distilled,  purified,  dried  and 
weighed.  A  determination  of  specific  gravity  shows  the  percentage 
of  methyl  iodide  in  the  mixture  and  from  this  the  percentage  of 
methyl  alcohol  in  the  original  sample.  Ethyl  alcohol  is  not  com- 
pletely transformed  into  iodide,  but  according  to  Lam  the  trans- 
formation of  the  methyl  alcohol  is  quantitative. 

The  Oxidation  Method  of  Thorpe  and  Holmes  ||  is  simpler  and  more 
direct.  It  depends  upon  the  oxidation  of  the  mixture  of  ethyl  and 
methyl  alcohols  under  such  conditions  that  the  former  is  converted 
into  acetic  acid  while  the  latter  is  completely  oxidized  to  carbon 
dioxide  and  water.  The  total  amount  of  alcohols  having  been  de- 
termined as  above,  a  part  of  the  distillate  is  mixed  with  water  in 
such  proportions  that  50  c.c.  of  the  mixture  shall  contain  not  more 
than  i  gram  of  methyl  alcohol  nor  more  than  4  grams  of  ethyl  and 
methyl  alcohols  together.  Fifty  c.c.  of  this  mixture  are  introduced 

*  Mulliken  and  Scudder  :  Amer.  Chem.  Journ.,  1900,  24,  444.     Prescott :  Pharma- 
ceutical Archives,  1901,  4,  86.     Bulletin  65,  Bur.  Chem.,  U.  S.   Dept.  Agriculture. 
f  Loc.  cit. 
JTrillat:   Compt.  rend.,  1899,  I.     Wolff:  Ztschr.  Unters.  Nahr.-Genussm.,  1901, 

4,  391- 

§  Ztschr.  angew.  Chem.,  1898,  125. 
\\Journ.  Chem.  Soc.,  1904,  85,  I. 


ALCOHOLS.  35 

into  a  300  c.c.  flask  having  a  tight  stopper  and  fitted  with  a  funnel 
and  side  tube,  20  grams  of  potassium  dichromate  and  80  c.c.  of 
dilute  sulphuric  acid  (1:4)  added  and  the  mixture  allowed  to  re- 
main for  1 8  hours.  A  further  quantity  of  10  grams  of  potassium 
dichromate  and  50  c.c.  of  sulphuric  acid  mixed  with  an  equal 
volume  of  water  are  now  added,  and  the  contents  of  the  flask 
heated  to  the  boiling  point  for  about  10  minutes,  the  evolved  car- 
bon dioxide  being  swept  out  of  the  apparatus  by  a  current  of  air 
and  collected  in  weighed  soda-lime  tubes.  Under  these  conditions 
each  gram  of  ethyl  alcohol  was  found  to  yield  about  o.oi  gram  of 
carbon  dioxide.  The  remaining  carbon  dioxide  found  is  calculated 
as  being  derived  from  the  complete  oxidation  of  methyl  alcohol. 
This  method  is  used  for  the  quantitative  determination  of  methyl 
alcohol  in  the  Government  Laboratory,  London. 

Amyl  Alcohols. 

The  amyl  alcohols  are  the  principal  constituents  of  fusel  oil,  and 
most  of  the  methods  proposed  for  the  determination  of  fusel  oil  in 
distilled  liquors  are  essentially  attempts  to  estimate  the  amyl  alco- 
hols. In  order  to  separate  the  amyl  alcohols  from  the  relatively 
large  amounts  of  ethyl  alcohol  ordinarily  present,  advantage  is 
taken  of  the  fact  that  the  former  are  much  more  soluble  in  chloro- 
form than  is  the  latter,  so  that  on  shaking  a  small  amount  of  chloro- 
form with  a  distillate  containing  about  30  per  cent,  of  alcohol, 
practically  all  of  the  amyl  alcohols  and  only  a  little  of  the  ethyl 
alcohol  pass  into  the  chloroform  layer.  The  amount  of  amyl  alco- 
hol removed  by  the  chloroform  is  usually  estimated  either  : 

1.  By  measuring  the  increase  in  volume  of  the  chloroform  layer 
(Roese's  method). 

2.  By  oxidizing  with  potassium  dichromate  and  determining  the 
amount  of  valerianic  acid  produced  (Marquardt's  method). 

Neither  method  gives  very  satisfactory  results.  The  Association 
of  Official  Agricultural  Chemists  *  has  adopted  the  Roese  method 
as  carried  out  by  Windisch.  Allen  f  recommends  a  modification 
of  the  Marquardt  method. 

METHODS  OF  STATING  STRENGTH  OF   ALCOHOL  SOLUTIONS. 
The  most  satisfactory  method  of  expressing  the  amount  of  alco- 
hol in  a  solution  is  in  terms  of  percentage  by  weight.     The  use  of 

*Bull.  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 
|  Commercial  Organic  Analysis,  Vol.  I,  p.   154. 


36  ORGANIC   ANALYSIS. 

the  "  percentage  by  volume  "  is,  however,  probably  more  common, 
while  frequently  the  results  of  an  analysis  of  alcoholic  liquor  are 
stated  in  grams  of  alcohol  per  100  c.c.  of  sample. 

For  revenue  purposes,  both  in  the  United  States  and  Great  Brit- 
ain, the  strength  of  alcoholic  liquors  is  expressed  in  terms  of 
"  proof  spirit,"  but  the  term  has  different  meanings  in  the  two 
countries.  American  proof  spirit  contains  50  per  cent,  of  alcohol 
by  volume,  corresponding  to  about  42.7  per  cent,  by  weight.  Brit- 
ish proof  spirit  is  defined  by  Parliament  as  having  such  a  density 
that  at  57°  F.,  thirteen  volumes  shall  weigh  the  same  as  twelve 
volumes  of  water  at  the  same  temperature.  This  corresponds  to 
about  49.2  per  cent,  by  weight. 

The  U.  S.  Pharmacopeia  authorizes  the  designation  "Absolute 
Alcohol"  for  that  which  contains  at  least  99  per  cent,  by  weight 
and  "  Alcohol"  for  that  containing  91  per  cent,  by  weight  or  94  per 
cent,  by  volume. 

"  Spirit"  of  the  German  Pharmacopeia  contains  about  86  per 
cent,  of  alcohol  by  weight. 

"  Rectified  Spirit"  of  the  British  Pharmocopeia  has  84  per  cent, 
by  weight  and  British  "Methylated  Spirit"  consists  of  nine  parts 
of  rectified  spirit  to  one  part  of  commercial  "wood  spirit"  or 
"  wood  naphtha,"  the  latter  containing,  according  to  recent  obser- 
vations of  Thorpe  and  Holmes,  from  72  to  80  per  cent,  of  methyl 
alcohol  by  volume. 

GLYCEROL. 

Glycerol  is  a  colorless,  odorless,  viscous  liquid  of  sweet  taste  and 
neutral  reaction,  miscible  in  all  proportions  with  water  and  with 
alcohol.  It  also  dissolves  in  mixtures  of  alcohol  and  ether,  but  is 
only  very  sparingly  soluble  in  pure  ether  *  and  practically  insoluble 
in  chloroform,  carbon  disulphide  and  benzene.  The  specific  gravity 
of  pure  glycerol  at  15°  referred  to  water  at  the  same  temperature, 
is  variously  stated  at  from  1.265  to  1.2677.  Anhydrous  glycerol 
boils  at  about  290°,  but  evaporates  rapidly  at  lower  temperatures 
(160°  or  over)  and  the  evaporation  is  greatly  accelerated  by  the 
presence  of  a  small  amount  of  water.  When  kindled,  glycerol 
burns  with  a  blue  flame  and  leaves  no  carbonaceous  residue. 

These  properties  together  with  the  fact  that  it  yields  acrolein 
when  heated  with  acid  potassium  sulphate,  are  usually  sufficient  for 

*  According  to  Lewkowitsch,  one  part  of  glycerol  of  1.23  sp.  gr.  dissolves  in  about 
500  parts  of  ether. 


ALCOHOLS.  37 

the  identification  of  glycerol  when  in  a  fairly  pure  and  concentrated 
state. 

Glycerol  is  a  good  solvent  for  many  substances,  both  organic 
and  inorganic,  and  its  presence  often  increases  their  solubility  in 
aqueous  and  alcoholic  solutions.  This  fact  and  the  difficulty  of 
distilling  without  loss  *  make  it  troublesome  to  separate  glycerol 
as  a  pure  aqueous  solution  as  is  done  in  the  determination  of  alco- 
hol. The  method  to  be  followed  in  removing  the  other  substances 
which  may  interfere  with  the  determination  will  depend  upon  the 
nature  of  the  sample.  The  final  determination  is  usually  made  in 
one  of  three  ways:  (i)  By  oxidation  with  potassium  dichromate. 
(2)  By  the  formation  of  triacetin.  (3)  By  separation  and  weighing. 

The  first  and  second  methods  will  be  described  as  applied  to 
crude  or  commercial  glycerins,  the  third  as  applied  to  wines. 

DETERMINATION  BY  OXIDATION. 

While  other  methods  of  determining  glycerol  by  oxidation  have 
been  proposed,  that  of  Hehner  |  is  the  one  now  generally  used. 
It  is  based  on  the  fact  that  glycerol  can  be  quantitatively  oxidized 
to  carbon  dioxide  and  water  by  heating  with  potassium  dichromate 
in  the  presence  of  sulphuric  acid. 

Reagents.  —  (i)  Strong  solution  of  potassium  dichromate  made 
by  dissolving  74.64  grams  of  the  pure  salt  (previously  pulverized 
and  dried  at  130°-!  35°)  and  diluting  the  solution  to  one  liter. 
(2)  Weak  solution  of  dichromate  made  by  diluting  100  c.c.  of  the 
strong  solution  to  one  liter.  (3)  A  solution  containing  about 
240  grams  of  ferrous  ammonium  sulphate  and  about  10  c.c.  of 
concentrated  sulphuric  acid  per  liter. 

Apparatus  which  is  to  come  in  contact  with  the  dichromate  solu- 
tion must  be  very  carefully  cleaned  in  advance  with  a  solution  of 
dichromate  in  strong  sulphuric  acid. 

Determination.  —  Weigh  such  an  amount  of  the  sample  as  will 
contain  about  1.5  grams  of  actual  glycerol  into  a  100  c.c.  flask, 
dilute  to  about  IO  c.c.,  add  0.5  to  i.o  gram  silver  oxide  and  let 

*  An  approximate  separation  of  glycerol  from  dissolved  solids  can  easily  be  made  by 
distilling  with  steam.  At  sufficiently  reduced  pressure  the  steam  distillation  may  be 
carried  on  with  very  little  loss  of  glycerol,  this  process  being  used  on  a  large  scale  as  a 
means  of  refining  commercial  glycerin.  The  necessary  apparatus  is,  however,  not 
always  available  in  the  laboratory  and  the  completeness  of  distillation  and  recovery  of 
the  glycerol  has  not  yet  been  definitely  settled. 

^Journ.  Soc.  Chem.  Ind.,  1889,  8,  4. 


38  ORGANIC   ANALYSIS. 

stand  for  ten  minutes,  shaking  frequently ;  then  add  basic  lead 
acetate  in  slight  excess  and  fill  to  the  mark.  After  thorough  shak- 
ing, filter  through  dry  paper,  discarding  the  first  few  c.c. ;  transfer 
25  c.c.  of  the  filtrate  to  beaker,  add  30  c.c.  of  a  mixture  of  equal 
volumes  of  sulphuric  acid  and  water  and  then  run  in  50  c.c.  of  the 
stronger  solution  of  potassium  dichromate,  measuring  the  volume 
with  the  greatest  possible  accuracy.  Cover  the  beaker  with  a 
watch  glass  and  place  it  in  boiling  water  for  two  hours.  During 
this  time  the  exact  strength  of  the  ferrous  sulphate  solution  is 
determined  by  titration  against  the  dichromate.  At  the  end  of  the 
two  hours  remove  the  beaker  from  the  boiling  water,  add  100  c.c. 
water  and  titrate  the  excess  of  dichromate  remaining.  Each  cubic 
centimeter  of  the  stronger  dichromate  solution  which  has  been  re- 
duced indicates  the  oxidation  to  carbon  dioxide  and  water  of  o.oi 
gram  of  glycerol. 

Notes.  —  The  treatment  of  the  sample  with  silver  oxide  pre- 
cipitates chlorides  and  oxidizes  any  aldehydes  which  may  be 
present.  Basic  lead  acetate  precipitates  proteids,  resinous  matter, 
and  the  higher  fatty  acids.  Hehner  supposed  that  these  two 
reagents  would  remove  all  impurities  likely  to  be  found  in  crude 
glycerins  which  could  be  oxidized  by  dichromate  under  the  con- 
ditions given.  In  a  number  of  cases,  however,  genuine  crude 
glycerins  have  given  high  results  by  this  method,  doubtless 
.because  they  contained  oxidizable  impurities  not  removed  by  silver 
oxide  and  basic  lead  acetate. 

As  the  oxidizing  dichromate  solution  is  a  strong  one,  it  must 
not  only  be  measured  with  the  greatest  care  but  it  should  always 
be  shaken  before  using  in  order  to  wash  down  any  drops  of  water 
which  may  have  evaporated  from  the  solution  and  condensed  on 
the  inner  surface  of  the  bottle.  Attention  must  also  be  paid  to  the 
temperature  at  the  time  of  measuring.  According  to  Hehner,  this 
solution  expands  0.05  per  cent,  for  each  increase  of  one  degree  C. 

As  the  method  is  ordinarly  described,  the  excess  of  dichromate 
remaining  after  the  oxidation  of  the  glycerol  is  reduced  by  adding  a 
known  amount  of  the  ferrous  ammonium  sulphate  solution  and  the 
excess  of  the  latter  titrated  by  the  weaker  dichromate.  The  green 
color  of  the  solution  makes  the  end  reaction  with  ferricyanide 
somewhat  difficult  to  detect  and  it  will  usually  be  found  more  satis- 
factory to  determine  the  excess  of  the  stronger  dichromate  by 
direct  titration  with  the  ferrous  solution.  Then  if  the  end  point 


ALCOHOLS.  39 

is  accidentally  overrun,  add  quickly  5  or  10  c.c.  of  the  weak  (equal 
to  0.5  or  i.o  c.c.  of  the  strong)  dichromate  and  continue  titrating 
with  the  ferrous  solution  until  the  blue  reaction  with  ferricyanide 

appears. 

DETERMINATION  BY  ACETYLATION. 

On  heating  with  acetic  anhydride,  glycerol  is  completely  con- 
verted into  the  triacetate.  This  is  the  basis  of  the  acetin  method 
of  Benedict  and  Cantor  *  which  is  applied  to  commercial  glycerins 
as  follows : 

Weigh  I  to  1.5  grams  of  the  sample  (which  must  not  contain 
more  than  30  to  40  per  cent,  of  moisture)  into  a  flask,  add  3  to  5 
grams  of  recently  fused  sodium  acetate  and  7  to  10  c.c.  acetic  an- 
hydride and  boil  under  a  reflux  condenser  for  one  and  one-half 
hours.  Allow  to  cool,  pour  50  c.c.  of  water  into  the  flask  through 
the  condenser  and  heat  gently  with  the  condenser  still  attached 
until  the  liquid  begins  to  boil  and  the  oily  layer  of  triacetate  goes 
into  solution.  Remove  the  condenser,  add  phenolphthalein  and 
carefully  neutralize  with  5  per  cent,  sodium  hydroxide  solution. 
Care  must  be  taken  to  avoid  any  excess  of  alkali  at  this  point  as 
the  triacetate  is  easily  hydrolyzed.  Shake  the  solution  well  to  avoid 
any  local  excess  of  alkali  during  the  neutralization,  and  stop  adding 
alkali  as  soon  as  the  solution  changes  to  a  reddish-yellow  color. 
If  the  solution  were  made  sufficiently  alkaline  at  this  point  to  show 
a  full  pink  color,  the  final  result  would  be  too  low. 

To  the  neutralized  solution  add  25  c.c.  of  a  10  per  cent,  solution 
of  sodium  hydroxide  (accurately  measured  by  a  pipette),  boil  for 
10  to  15  minutes  and  titrate  the  excess  -of  alkali  with  accurately 
standardized  acid.  At  the  same  time  determine  the  exact  strength 
of  the  alkali  solution  by  measuring  out  another  portion  of  25  c.c. 
with  the  same  pipette  and  titrating  with  the  same  acid,  using 
phenolphthalein  as  indicator.  The  difference  between  the  two 
titrations  shows  the  amount  of  alkali  consumed  in  saponifying  the 
triacetate  and  from  this  the  amount  of  glycerol  is  calculated,  one 
molecule  of  glycerol  being  equivalent  to  three  molecules  of  sodium 
hydroxide. 

Notes.  —  If  sufficient  care  is  taken  to  prevent  any  of  the  acetin 
from  being  lost  by  evaporation  or  saponified  during  the  neutraliza- 
tion of  the  excess  of  acetic  anhydride,  the  results  by  this  method 
are  very  accurate.  If  these  precautions  are  not  carefully  observed 

*  Ztschr.  angew.  Ghent.,  1 888,  460. 


40  ORGANIC   ANALYSIS. 

the  results  are  likely  to  be  somewhat  low.  Practically  pure  glycerol 
should  give  the  same  results  by  acetylation  as  by  oxidation.  Crude 
glycerins  often  give  much  higher  results  by  the  latter  method.  In 
such  cases  the  acetin  method  should  be  repeated  with  special 
attention  to  the  precautions  already  mentioned  and  to  the  moisture 
present  in  the  sample  and  the  sodium  acetate  used.  Unless  the 
sample  contains  over  60  per  cent,  of  glycerol  and  the  sodium 
acetate  is  strictly  anhydrous,  the  formation  of  the  triacetate  is  likely 
to  be  incomplete.  When  obtained  under  the  proper  conditions, 
the  results  by  the  acetin  method  should  be  preferred  to  those  by 
the  dichromate  because  the  impurities  of  crude  glycerins  are  more 
likely  to  vitiate  the  latter  than  the  former  method.  In  cases  where 
other  oxidizable  substances  are  absent  or  can  be  entirely  removed, 
it  is  a  good  plan  to  make  a  determination  by  each  method  and 
average  the  results  obtained.  For  dilute  solutions  the  dichromate 
method,  if  applicable,  is  much  more  convenient  because  the  acetin 
method  cannot  be  applied  until  after  the  glycerol  has  been  concen- 
trated by  careful  evaporation. 

DETERMINATION  BY  SEPARATION  AND  WEIGHING. 

For  the  estimation  of  glycerol  in  fermented  liquors  where  it 
occurs  mixed  with  relatively  large  amounts  of  alcohol,  sugar,  or- 
ganic acids  and  resinous,  nitrogenous  and  other  "  extractive" 
matters,  the  plan  usually  followed  is  to  evaporate  off  the  greater 
part  of  the  alcohol  and  water  and  then  treat  with  lime  and  after 
further  evaporaton  take  up  the  glycerol  with  strong  alcohol,  leaving 
the  greater  part  of  the  extractive  matters  in  the  residue.  The  sub- 
stances, other  than  glycerol,  which  pass  into  the  alcohol  solution 
are  largely  precipitated  by  the  addition  of  ether  and  the  alcohol- 
ether  solution  is  then  evaporated. 

The  details  of  the  process  as  adopted  by  the  Association  of 
Official  Agricultural  Chemists  *  for  the  determination  of  glycerol 
in  wine  are  as  follows : 

Evaporate  100  c.c.  of  wine|  in  a  porcelain  dish  on  the  water 

*Bull.  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

f  With  wines  whose  extract  exceeds  5  grams  per  100  c.c.,  heat  to  boiling  in  a  flask 
the  portion  to  be  used  in  the  determination  of  glycerol,  and  treat  with  successive  small 
portions  of  milk  of  lime  until  it  becomes,  first,  darker,  and  then  light  in  color.  When 
cool,  add  200  c.c.  of  96  per  cent,  alcohol  (sp.  gr.  0.812),  allow  the  precipitate  to 
subside,  filter,  and  wash  with  96  per  cent,  alcohol.  Evaporate  the  filtrate  to  about 
10  c.c. ,  add  about  5  grams  of  sand  and  from  1.5  to  2  c  .c.  of  milk  of  lime,  and  proceed 
as  directed  above. 


ALCOHOLS.  41 

bath  to  a  volume  of  about  10  c.c.  and  treat  the  residue  with  about 
5  grams  of  fine  sand  and  with  from  1.5  to  2  c.c.  of  milk  of  lime 
(containing  about  40  per  cent,  of  calcium  hydroxide  or  30  per  cent, 
of  calcium  oxide)  for  each  gram  of  extract  present,  and  evaporate 
almost  to  dryness.  Treat  the  moist  residue  with  5  c.c.  of  96  per 
cent,  alcohol  (sp.  gr.  0.812),  remove  the  substance  adhering  to  the 
sides  of  the  dish  with  a  spatula,  and  rub  the  whole  mass  to  a  paste, 
with  the  addition  of  a  little  more  alcohol.  Heat  the  mixture  on 
the  water  bath,  with  constant  stirring,  to  incipient  boiling,  and 
decant  the  liquid  into  a  flask  graduated  at  looand  HO  c.c.  Wash 
the  residue  repeatedly  by  decantation  with  10  c.c.  portions  of  hot 
96  per  cent,  alcohol.  Cool  the  contents  of  the  flask  to  15°,  dilute 
to  the  no  c.c.  mark  with  96  per  cent,  alcohol,  and  filter  through  a 
folded  filter.  Evaporate  100  c.c.  of  the  filtrate  to  a  sirupy  con- 
sistency in  a  porcelain  dish  on  a  hot,  but  not  boiling,  water  bath, 
transfer  the  residue  to  a  small  glass-stoppered  graduated  cylinder 
with  20  c.c.  of  absolute  alcohol,  and  add  three  portions  of  10  c.c. 
each  of  absolute  ether,  mixing  after  each  addition.  Let  stand  until 
clear,  then  pour  off  through  a  filter,  and  wash  the  cylinder  and 
filter  with  a  mixture  of  one  part  absolute  alcohol  to  one  and  one- 
half  parts  ot  absolute  ether,  pouring  the  wash  liquor  also  through 
the  filter.  Evaporate  the  filtrate  to  a  sirupy  consistency,  dry  for 
one  hour  at  the  temperature  of  boiling  water,  weigh,  ignite,  and 
weigh  again.  The  loss  on  ignition  increased  by  one-tenth  gives 
the  glycerol  expressed  in  grams  per  IOO  c.c. 

Notes.  —  When  a  mixture  of  glycerol  and  water  is  concentrated 
at  about  the  boiling  point  of  the  latter,  water  evaporates  without 
any  considerable  loss  of  glycerol  until  the  latter  becomes  quite 
highly  concentrated.  The  point  at  which  glycerol  begins  to  be 
lost  depends  upon  a  number  of  conditions  such  as  temperature,  the 
depth  of  the  layer,  etc.,  but  some  loss  always  occurs  in  expelling 
the  last  of  the  water.  The  same  is  true  with  regard  to  the  concen- 
tration of  alcoholic  solutions  though  the  loss  here  is  less  as  glycerol 
is  less  volatile  with  alcohol  than  with  water  vapor.  On  the  other 
hand  the  glycerol  obtained  by  this  method  has  other  impurities 
than  ash,  and  these  are  calculated  as  glycerol.  For  detailed  tests 
of  the  method  see  the  work  of  Neubauer  and  Borgmann  on  wines  * 
and  that  of  Clausnitzer  on  beers.f 

*  Ztschr.  anal.  Chem.,  1878,  17,  442. 

t/Jftf.,  1881,20,  58. 

See  also  — Friedeberg  :  Ueber  Glycerinbestimmung  in  vergohrenen  Getranken,  Dis- 


42  ORGANIC   ANALYSIS. 

EXAMINATION  OF  COMMERCIAL  GLYCEROL. 

Commercial  glycerol  (or  "  glycerin"  )  always  contains  water,  and 
all  but  the  best  "chemically  pure  glycerol"  contains  appreciable 
amounts  of  impurities  derived  from  the  original  fat  or  from  the 
saponifying  agents.  The  tests  to  be  applied  and  the  allowable 
limits  of  impurity  will  depend  upon  the  purpose  for  which  the  gly- 
cerin is  to  be  used.  The  quantitative  determinations  most  com- 
monly required  are :  specific  gravity,  carbonaceous  residue,  ash 
and  salt.  The  higher  grades  of  glycerin  must  also  be  practically 
neutral  and  show  no  turbidity  due  to  fatty  acids  when  the  sample 
is  diluted  with  water,  and  hydrochloric  acid  added. 

Additional  tests  and  specifications  for  pharmaceutical  glycerin 
will  be  found  in  the  U.  S.  Pharmacopeia  and  Dispensatory. 

Glycerin  used  for  the  manufacture  of  nitroglycerol  need  not  be 
colorless  but  must  have  a  high  degree  of  purity  as  shown  by  the 
following  quantitative  determinations. 

Specific  Gravity. 

The  viscosity  of  glycerol  and  the  fact  that  when  highly  concen- 
trated it  is  quite  hygroscopic  make  it  necessary  to  use  the  greatest 
care  in  this  determination.  Hehner*  recommends  the  Sprengel 
tube  and  Lewkowitsch  |  the  ordinary  specific  gravity  bottle.  It 
is  usually  more  convenient  to  take  the  specific  gravity  by  using 
the  hydrostatic  sinker  with  the  analytical  balance.  For  this  pur- 
pose the  Reimann  sinker  graduated  for  15°  is  very  convenient. 
The  glass  body  encloses  a  thermometer  (usually  graduated  from  o° 
to  27°)  and  is  of  such  size  that  when  immersed  it  displaces  5  (or 
sometimes  10)  grains  of  water  at  15°.  This  calibration  should  be 
tested  for  each  sinker  as  it  is  received  from  the  dealer  by  suspend- 
ing it  over  the  pan  of  the  analytical  balance,  finding  its  weight 
in  air  and  then  determining  its  weight  when  suspended  in  the  same 
position  but  immersed  in  recently  boiled  distilled  water  at  15°. 
This  is  easily  done  by  placing  a  bridge  over  the  balance  pan  and 
on  this  bridge  a  small  cylinder  containing  enough  water  to  cover 
the  sinker  to  a  depth  of  about  I  cm.  The  sinker  should  hang 
in  the  center  of  the  cylinder  and  the  diameter  of  the  latter  should 

sertation,  Berlin,  1890.     Schaumann  :  Bestimmung  von  Glycerin  im  Wein,  Disserta- 
tion, Erlangen,  1891. 

*Journ.  Soc.  Chem.  Ind.,  1889,  8,  4. 

fOils,  Fats  and  Waxes  [3d  Ed.],  p.  1104. 


ALCOHOLS. 


43 


be  about  three  times  that  of  the  former.  Having  found  the  weight 
of  water  displaced,  remove  and  dry  the  sinker,  replace  the  cylin- 
der of  water  by  one  filled  to  the  same  depth  with  the  sample  of 
glycerin  to  be  tested  and  weigh  the  sinker  suspended  in  the  glyc- 
erin at  15°.  The  loss  of  weight  in  glycerin  divided  by  that  in 
water  shows  the  specific  gravity  of  the  former  referred  to  water  at 
the  same  temperature. 

If  the  sample  consists  of  glycerol  and  water  with  only  insignifi- 
cant amounts  of  impurities,  the  specific  gravity  serves  as  a  meas- 
ure of  the  concentration.  This  method  is,  however,  considerably 
less  accurate  than  in  the  determination  of  alcohol  because  the 
specific  gravity  of  pure  anhydrous  glycerol  is  less  accurately 
known  than  that  of  absolute  alcohol.  The  most  generally  ac- 
cepted tables  of  specific  gravities  of  glycerol  solutions  are  those  of 
Lenz*  and  Nicol.f  Reduced  to  15.5°  and  15°,  the  values  for  con- 
centrated solutions  as  found  by  these  observers  are  : 


Glvcerol 
Percent. 

Specific  Gravity 
at  15.5°  Lenz. 

Specific  Gravity 
at  15°  Nicol. 

Glycerol 
Percent. 

Specific  Gravity 
at  15.5°  Lenz. 

Specific  Gravity 
at  15°  Nicol. 

100 

.2674 

.26596 

87 

.2327 

.23128 

99 

.2647 

•26335 

86 

.2301 

.22855 

98                    3 

.2620 

.26072 

85 

•2274 

.22583 

97 

•2594 

.25809 

84 

.2248 

.22310 

96 

.2567 

.25547                      83 

.2222 

.22038 

95 

.2540 

.25285                      82 

.2196 

.21766 

94 

•2513 

.25021 

81 

.2169 

•21493 

93 

.2486 

.24756 

80 

•2143 

.21221 

92 

.2460 

.24487 

79 

.2117 

.20949 

91 

•2433 

.24217 

78 

.2090 

.20677 

9° 

.2406 

.23945 

77 

.2064 

.  20404 

89 

.2380 

•23673 

76 

•2037 

.20131 

88 

•2353 

.23400 

75 

.2011 

.19857 

It  has  also  been  proposed  to  apply  a  correction  for  the  influence 
of  salt  on  the  specific  gravity  and  use  the  corrected  figure  as  a 
rough  measure  of  the  glycerol  present  in  crude  glycerins.J 

If  the  percentage  of  glycerol  indicated  by  the  specific  gravity  is 
less  than  that  shown  by  the  dichromate  method,  the  presence  of 
oxidizable  compounds  of  lower  density  than  glycerol  is  indicated. 
Thus  a  sample  of  "  light "  glycerin  examined  by  Noyes  and  Wat- 
kins  §  was  found  to  contain  about  38  per  cent,  of  trimethylene 

*  Recommended  by  Hehner,  Journ.  Soc.  Chem.  Ind.y  1889,  8,  4. 
fU.  S.  Dispensatory  (i8th  Ed.),  p.  657. 

J  Richardson  and  Joffe,  Journ.  Soc.  Chem.  Ind.,  1898,  17,  330. 
fy  Journ.  Amer.  Chem.  Soc.,  1895,  17,  890. 


44  ORGANIC   ANALYSIS. 

glycol.  Since  this  glycerin  was  obtained  largely  from  refuse  house 
fat,  the  glycol  was  probably  formed  by  spontaneous  saponification 
and  subsequent  fermentation  of  the  glycerol  in  the  impure  rancid 
fat.  This  glycol  has  also  been  found  in  considerable  quantity  in 
the*' tank  liquor"  separating  from  the  fat  before  saponification* 
and  is  probably  not  an  uncommon  impurity,  especially  in  glycerin 
obtained  from  refuse  fats. 

Samples  containing  glycol  would,  of  course,  yield  lower  results 
by  the  acetin  than  by  the  dichromate  method. 

Carbonaceous  Residue  and  Ash. 

Weigh  about  50  grams  of  glycerin  into  a  50  c.c.  platinum  dish, 
heat  cautiously  over  a  Bunsen  burner  until  the  sample  ignites, 
remove  the  burner  and  allow  the  glycerol  to  burn  away,  being 
careful  that  the  dish  is  not  exposed  to  draughts.  When  burning 
ceases,  transfer  the  dish  at  once  to  a  desiccator  and  when  cool 
weigh  quickly,  as  the  residue  is  apt  to  be  hygroscopic.  After 
weighing  the  carbonaceous  residue,  burn  to  whiteness  and  weigh 
the  ash. 

Sodium  Chloride. 

Weigh  50  to  100  grams  of  sample,  dilute  with  twice  its  volume 
of  water,  add  a  few  drops  of  potassium  chromate  solution  and 
titrate  with  standard  silver  nitrate,  preferably  tenth-normal  or  of 
such  strength  as  to  be  equivalent  to  a  one  per  cent,  solution  of  so- 
dium chloride. 

Specifications  for  Dynamite  Glycerin. 

The  following  specifications  are  furnished  by  the  firm  of  Wm. 
F.  Jobbins  as  representing  the  requirements  of  a  majority  of  the 
nitroglycerol  manufacturers  in  the  United  States  at  the  present 
time  (1904). 

Specific  Gravity  at  15°,  not  less  than  1.2625. 

Carbonaceous  Residue  (including  ash)  and  Ash  as  determined  by 
the  method  given  above,  must  not  exceed  0.03  per  cent,  and  o.oi 
per  cent,  respectively. 

Sodium  Chloride  determined  as  above  must  not  exceed  o.oi  per 
cent,  and  is  usually  very  much  less  than  this  amount. 

Total  Acid  Equivalent  ("  Fatty  Acids  ")  is  determined  as  follows : 
Dilute  the  sample  with  twice  its  volume  of  water,  add  a  few  drops 
of  phenolphthalein  solution  and  a  decided  excess  of  standard 

*Twitchell:  Journ.  Amer.  Chen*.  Soc.,  1895,  17,  891. 


ALCOHOLS.  45 

sodium  hydroxide;  boil  five  minutes  and  then  titrate  the  excess  of 
alkali  by  standard  acid.  The  glycerin  must  not  consume  more 
than  o.io  per  cent,  of  its  weight  of  sodium  hydroxide  in  this  test. 

Fora  fuller  discussion  of  the  examination  of  "  dynamite  glycerin," 
see  a  paper  by  Barton  in  the  Jomnal  of  the  American  Chemical 
Society  (\%9l,  17,  277). 

Detailed  information  on  the  testing  of  "  dynamite  glycerin " 
and  other  grades  of  commercial  glycerol  will  be  found  in  Lewko- 
witsch's  "Oils,  Fats  and  Waxes,"  and  in  Allen's  "Commercial 
Organic  Analysis." 


CHAPTER  IV. 

Aldehydes . 

The  most  important  methods  for  the  detection  and  determination 
of  aldehydes  are  based  upon  reactions  of  oxidation,  of  condensation 
and  of  direct  addition. 

The  readiness  with  which  aldehydes  undergo  oxidation  gives 
them  the  property  of  reducing  ammoniacal  silver  solution,  which 
is  the  basis  of  one  of  the  most  delicate  qualitative  tests  for  this 
group  of  compounds.  The  test  may  be  carried  out  as  follows  :  * 

Mix,  in  a  test-tube  previously  cleaned  with  hot  sodium  hydrox- 
ide solution,  i  c.  c.  of  ammoniacal  silver  nitrate  solution  (con- 
taining one  part  of  silver  nitrate  in  ten  parts  of  ammonium  hydrox- 
ide of  0.923  sp.  gr.)  and  I  c.c.  of  ten  per  cent  sodium  hydroxide 
solution.  Shake  the  mixture  in  the  tube  and  then  allow  two  or 
three  drops  of  the  solution  to  be  tested  to  flow  slowly  down  the 
moistened  glass  surface  into  the  reagent.  Shake  and  allow  to 
stand  cold  for  five  minutes.  Aldehydes  (and  a  few  other  com- 
pounds including  some  of  the  polyatomic  alcohols)  cause  the  pro- 
duction of  a  dark  brown  or  black  precipitate  or  mirror  of  metallic 
silver.  This  reaction  is  given  by  all  of  the  ordinary  aldehydes  of 
the  fatty  series,  including  the  aldose  carbohydrates,  but  not  by  all 
aromatic  aldehydes. 

The  ammoniacal  silver  solution  and  the  sodium  hydroxide 
must  not  be  mixed  in  advance  and  must  always  be  kept  cool  as  a 
dangerously  explosive  precipitate  is  apt  to  form  on  warming  or  on 
long  standing.  The  use  of  a  mixture  of  sodium  hydroxide  and 
ammoniacal  silver  nitrate  (Tollens*  aldehyde  reagent)  makes  the 
test  more  delicate  than  when  the  ammoniacal  silver  solution  is 
used  alone. 

Alkaline  solutions  of  other  metals  are  reduced  by  many  aldehy- 
des, especially  on  boiling,  and  many  quantitative  methods  for  in- 
dividual aldehydes  are  based  upon  the  determination  of  the 
amount  of  metal  reduced. 

Condensation  reactions,  especially  with  phenylhy drazine,  hy  drox- 

*  Noyes  and  Mulliken  :  Identification  and  Class  Reactions  of  Organic  Substances. 
Mulliken :  Identification  of  Pure  Organic  Compounds,  Vol.  I.,  p.  22. 

46 


ALDEHYDES.  47 

ylamine  and  phenols,  are  often  used  for  the  detection  and  some- 
times for  the  determination  of  aldehydes.  A  general  discussion 
of  such  methods  will  be  found  in  the  works  of  Vaubel  *  and  of 
Meyer,  f  Several  special  methods  will  be  described  in  this  and 
the  two  following  chapters. 

Of  the  addition  reactions  of  aldehydes,  that  with  bisulphite  is  of 
especially  wide  application.  On  shaking  a  liquid  aldehyde  or  a 
concentrated  solution  of  aldehyde  in  water  or  ether,  with  an  equal 
volume  of  strong  sodium  bisulphite  solution,  addition  takes  place 
with  the  formation  of  the  saturated  compound  RCH(OH)SO3Na 
which  usually  separates  as  a  white  crystalline  precipitate.  Ketones 
containing  the  CH3CO  group  also  give  the  reaction.  A  nega- 
tive result  is  not  conclusive  as  the  addition  product  may  be  too 
soluble  to  appear  as  a  precipitate. 

According  to  Ripper,;):  the  bisulphite  reaction  can  be  utilized  for 
the  determination  of  any  aldehyde  soluble  in  water  or  which  can 
be  brought  into  solution  by  a  small  amount  of  alcohol.  A  one-half 
per  cent,  solution  of  the  aldehyde  is  mixed  with  twice  its  volume 
of  a  solution  of  potassium  bisulphite  of  known  strength  (about  12 
grams  per  liter),  and  after  1 5  minutes  the  excess  of  bisulphite  is 
determined  by  titration  with  iodine.  Ripper  applied  this  method 
with  satisfactory  results  to  solutions  of  formaldehyde,  acetaldehyde, 
benzaldehyde  and  vanillin. 

A  similar  addition  reaction  gives  rise  to  the  well-known  and 
delicate  "  fuchsin  test "  for  aldehydes.  This  test,  as  developed  by 
Mulliken,§  is  as  follows  : 

To  prepare  the  fuchsin  aldehyde  reagent,  dissolve  0.2  gram  of 
rosanilin,  or,  if  the  free  base  cannot  be  obtained,  of  the  hydro- 
chloride  or  acetate  in  10  c.c.  of  a  freshly  prepared,  cold,  saturated 
aqueous  solution  of  sulphur  dioxide.  Allow  the  solution  to  stand 
until  all  signs  of  pink  disappear  and  it  becomes  colorless  or  pale 
yellow.  This  will  require  several  hours.  Then  dilute  with  water 
to  200  c.c.  and  preserve  for  use  in  a  tightly-stoppered  bottle. 

To  5  c.c.  of  this  reagent  add  0.05  gram,  or  one  drop,  of  the  sub- 
stance to  be  tested  (if  pure,  or  a  few  drops  if  in  solution).  If  the 
substance  is  a  liquid,  or  dissolves  in  the  reagent,  allow  to  stand  two 

*Bestimmung  organische  Verbindungen. 

f  Analyse  und  Konstitutionsermittlung  organischer  Verbindungen. 

\Monatsh,  Chem.,  1900,  21,  1079. 

g  Identification  of  Pure  Organic  Compounds,  Vol.  I,  p.  15. 


48  ORGANIC   ANALYSIS. 

minutes  and  observe  the  color.  If  the  substance  does  not  dissolve, 
shake  gently  for  two  minutes  and  then  observe  the  color.  The 
appearance  of  a  distinct  pink,  red,  purple  or  blue  coloration  indi- 
cates the  presence  of  an  aldehyde.  The  test  to  be  of  value  must 
be  applied  under  carefully  regulated  conditions.  The  reagent  is 
reddened  by  alkalies  or  alkaline  salts  of  weak  acids,  by  heating  or 
by  long  exposure  to  air  at  ordinary  temperature.  In  general,  the 
test  as  here  described  distinguishes  aldehydes  other  than  carbohy- 
drates from  the  latter  and  from  ketones.  A  few  acetals  show  the 
reaction  through  being  partially  hydrolyzed  to  aldehydes  under  the 
conditions  of  the  test.  Acetone  and  some  other  soluble  ketones 
prepared  by  destructive  distillation  gradually  redden  the  reagent  if 
added  to  it  in  large  quantity  or  if  allowed  to  remain  in  contact  for 
a  number  of  minutes;  but  this  is  thought  to  be  due  chiefly,  if 
not  wholly,  to  the  presence  of  traces  of  aldehydes  or  acetals 
(Mulliken). 

This  reaction  serves  for  the  detection  of  minute  quantities  of  alde- 
hydes present  as  impurities  in  commercial  alcohol,  and  for  the  col- 
orimetric  estimation  of  aldehydes  in  distilled  liquors.* 

FORMALDEHYDE. 

Formaldehyde  gas,  produced  by  the  partial  oxidation  of  methyl 
alcohol,  is  freely  soluble  in  water  and  is  most  commonly  handled 
as  a  35  to  40  per  cent,  aqueous  solution.  Such  solutions  are  often 
designated  formalin,  formol  or  formal.  More  dilute  solutions  are 
extensively  sold  as  food  preservatives,  often  under  fanciful  or  mis- 
leading names. 

In  dilute  aqueous  solution,  formaldehyde  exists  in  the  "mono- 
molecular  "  state,  as  CH2O.  Such  solutions  do  not  change  if 
kept  at  ordinary  temperature  in  closed  vessels.  When  an  aqueous 
solution  is  concentrated  either  by  spontaneous  evaporation  or 
by  heating,  a  white  flocculent  deposit  appears.  If  the  solution  is 
then  separated  from  the  deposit  it  is  found  to  contain  condensed 
or  polymerized  formaldehyde. t  The  material  which  deposits  from 
a  concentrated  aqueous  solution  of  formaldehyde  has,  after  drying, 

*Medicus  :  Forschungsber.  iiber  Lebensmiltel,  1895,  J>  299  »  Bulls.  46  and  65,  Bur. 
Chem.,  U.  S.  Dept.  Agriculture. 

f  Tollens  and  Mayer:  Ber.  deut.  chem.  Ges.,  1888,  21,  1571,  3503.  Kraut,  Esch- 
weiler  and  Grossmann  :  Ann.  Chem.,  1890,  258,  103. 


ALDEHYDES.  49 

the  composition  (CH2O)6-H2O  to  (CH2O)8-H2O.*  It  is  amorphous, 
soluble  in  warm  water  and  has  an  odor  resembling  that  of  formalde- 
hyde. The  paraformaldehyde  of  commerce  consists  essentially  of 
this  material. 

Metaformaldehyde(oxymethylene,"trioxymethylene''),(CH2O)x, 
may  also  be  formed  by  evaporation  of  formaldehyde  solutions. 
By  prolonged  digestion  at  ordinary  temperature  or  by  heating  for 
a  short  time  at  I3O°-I5O°  with  a  large  excess  of  water,  metafornv 
aldehyde  passes  into  solution  and  into  the  "mono-molecular" 
form.  Polymeric  modifications  of  formaldehyde  in  aqueous  solu- 
tion resemble  closely  the  original  substance  in  its  behavior  toward 
reagents  so  that,  as  measured  by  the  ordinary  methods,  a  solution 
does  not  lose  strength  by  the  partial  polymerization  of  the  formal- 
dehyde so  long  as  all  remains  in  solution. 

Commercial  solutions  of  formaldehyde  commonly  contain 
methyl  alcohol  and  may  contain  any  of  the  impurities  of  commer- 
cial wood  spirit.  Solutions  of  the  usual  strength,  35  to  40  per 
cent.,  should  have  specific  gravities  of  about  1. 08  to  i.n  at  15°, 
lower  figures  ordinarily  indicating  the  presence  of  excessive 
amounts  of  methyl  alcohol.")* 

The  methods  given  in  this  chapter  for  the  detection  and  deter- 
mination of  formaldehyde  refer  especially  to  the  examination  of 
commercial  solutions  containing  only  such  impurities  as  ordinarily 
occur  in  crude  preparations  of  formaldehyde,  or  substances  which 
might  be  used  with  formaldehyde  in  preservative  mixtures.  The 
examination  of  food  products  for  formaldehyde  will  be  discussed 
in  connection  with  other  food  preservatives  in  a  subsequent 
chapter. 

If  a  solution  to  be  examined  contains  dissolved  solids  which 
interfere  with  the  direct  application  of  the  tests  as  described,  it 
can  be  acidified  with  a  small  excess  of  phosphoric  or  sulphuric  acid, 
distilled,  and  the  test  applied  to  the  distillate.  The  latter,  however, 
will  never  contain  all  of  the  formaldehyde,  since  some  is  always 
polymerized  and  left  as  paraformaldehyde  in  the  distilling  flask. 

*L6sekann:  Chem.  Zfg.,  1890,  14,  1408.  Delephine:  Compt.  rend.,  1897,  124, 
1525  ;  Beilstein  :  Organische  Chemie,  Erganzbd.,  I.,  467. 

|On  the  determination  of  methyl  alcohol  in  formaldehyde  solutions  see — Duyk: 
Ann,  Chim.  Anal.  AppL,  1901,  6,  407  ;  Journ.  Chem.  Soc.,  1902,  82,  ii,  no.  Stritrar  : 
Zfschr.  ana/.  Ghent.,  1904,  43,  401.  Gnehm  and  Kaufler  :  Ztschr.  angew.  Chem., 
1904,  17,  673;  1905,  18,  93.  Bamberger:  Ibid.,  1904,  17,  1246. 


50  ORGANIC  ANALYSIS. 

DETECTION  AND  IDENTIFICATION. 

Resorcin  Test* 

Mix  one  drop  of  a  I  per  cent,  aqueous  solution  of  resorcin  with 
I  c.c.  of  a  dilute  aqueous  solution  (preferably  about  0.2  per  cent.) 
of  the  aldehyde.  Allow  the  mixture  to  flow  gently  down  the  side 
of  an  inclined  test-tube  containing  3-5  c.c.  of  pure  concentrated 
sulphuric  acid  (or  incline  the  test-tube  containing  the  mixture  and 
pour  in  the  acid).  Impart  a  gentle  rotary  motion  to  the  liquids  by 
cautiously  swaying  the  lower  end  of  the  tube  through  a  circle 
about  a  decimeter  in  diameter,  in  such  a  manner  as  not  to  cause 
the  disappearance  of  the  two  layers.  If  formaldehyde  is  present, 
a  red  ring  slightly  tinged  with  violet  will  soon  appear.  Above 
this  ring  a  light  flocculent  precipitate,  at  first  nearly  white  on  its 
upper  surface  and  red-violet  beneath,  but  soon  changing  to  flocks 
that  are  red  throughout,  will  be  seen  suspended  in  the  aqueous 
upper  layer. 

This  reaction  is  very  satisfactory  for  solutions  containing  one 
part  of  formaldehyde  in  100  to  5000  parts  of  solution  and  can 
be  detected  to  a  dilution  of  I  :  100,000.  A  similar  reaction  is 
obtained  if  phenol  is  used  instead  of  resorcin. 

Gallic  Acid  Test.  | 

Mix  0.2  c.c.  of  a  saturated  solution  of  gallic  acid  in  pure  ethyl 
alcohol,  with  I  to  2  c.c.  of  the  solution  to  be  tested  and  introduce 
a  layer  of  concentrated  sulphuric  acid,  as  in  the  resorcin  test.  In 
the  presence  of  formaldehyde,  a  green  zone  appears  at  the  line  of 
contact  of  the  two  liquids.  This  gradually  changes  to  a  pure  blue 
ring,  which,  in  the  case  of  pure  aqueous  solutions  of  formaldehyde, 
can  be  detected  without  difficulty  at  a  dilution  of  I  :  500,000.  If 
the  solution  tested  contains  as  much  as  one  part  of  formaldehyde 
in  20,000,  a  yellowish  color  appears  immediately  at  the  line  of  con- 
tact of  the  two  liquids.  This  quickly  turns  green,  and  the  blue  color 
develops  both  above  and  below  the  green  zone.  If  other  substances 
which  give  color  reactions  are  also  present,  the  upper  layer  will  vary 
in  color,  but  the  green  and  lower  blue  ring  will  still  appear  beneath 

*  Mulliken  and  Scudder:  Amer.  Chem.  Journ.,  1900,  24,  451.  Mulliken  :  Iden- 
tification of  Pure  Organic  Compounds,  Vol.  I,  p.  24. 

f  Barbier  and  Jandrier  :  Ann.  Chim.  Anal.  AppL,  i,  325;  Abs.  Analyst,  1896, 
21,  295.  Mulliken  and  Scudder  :  Amer.  Chem.  Journ.,  1900,  24,  446. 


ALDEHYDES.  5* 

(Mulliken  and  Scudder).  On  swaying  the  tube,  or  allowing  it  to 
stand  for  some  time,  the  blue  color  spreads  throughout  the  zone 
and  a  pure  blue  ring  is  usually  obtained.  The  color  is  quite  per- 
manent and  apparently  quite  characteristic,  no  other  substance 
having  been  noted  as  giving  the  blue  ring.  Acetaldehyde  tested 
in  the  same  way  gives  a  reddish  brown  coloration. 

Hydrochloric  Acid  and  Casein  Test.* 

Mix  5  c.c.  of  the  solution  to  be  tested  with  5  c.c.  of  pure  milk  in 
a  porcelain  casserole,  add  10  c.c.  of  concentrated  hydrochloric  acid 
containing  0.002  gram  of  ferric  chloride  and  heat  slowly  over  a 
free  flame  nearly  to  boiling,  meanwhile  giving  the  casserole  a 
rotary  motion  to  break  up  the  curd.  A  violet  coloration  indi- 
cates formaldehyde.  According  to  Leach,  various  aldehydes  give 
color  reactions  under  this  treatment,  but  formaldehyde  alone 
shows  the  unmistakable,  violet  coloration.  This  test  is  especially 
useful  for  the  detection  of  formaldehyde  in  milk,  and  will  be  more 
fully  discussed  in  that  connection. 

Methylene-di-ft-  naphthol  Test. 

Since  formaldehyde  is  frequently  sold  under  other  names,  its 
identification  by  some  method  independent  of  the  above  color  reac- 
tions may  be  a  matter  of  importance.  In  such  cases  the  following 
test  given  by  Mulliken  f  will  be  useful. 

Place  in  a  test-tubes  drops  of  a  30  to  40  per  cent,  or  10  drops  of 
a  10  per  cent,  solution,  3  c.c.  of  dilute  alcohol  (i  :  2),  0.04  to  0.06 
gram  /9-naphthol,  and  3  to  5  drops  of  concentrated  hydrochloric 
acid.  Boil  gently  until  the  liquid  becomes  filled  with  an  abundant 
precipitate  of  small  white  needles.  Filter  while  hot.  Wash  with 
I  c.c.  of  dilute  alcohol  (1:2).  Boil  the  precipitate  with  4  c.c.  of 
dilute  alcohol  (i  :i.)  (It  is  not  necessary  that  all  should  dissolve) 
Cool  and  filter  off  the  precipitate.  Wash  with  I  c.c.  of  dilute 
alcohol  (i  :  I.)  Dry  on  porous  tile  in  a  warm  place  and  determine 
the  melting  point. 

Methylene-di-/3-naphthol,  the  product,  forms  white  needles 
which,  when  the  temperature  in  the  neighborhood  of  the  melting 
point  is  raised  at  the  rate  of  i°  in  15  seconds,  begin  to  turn  brown 

*  Leach  :   Ann.    Kept.    Mass.     State  Board  of    Health  1897,   558;    1899,    699. 
Food  Inspection  and  Control  (New  York,  1904),  p.  140. 
f  Identification  of  Pure  Organic  Compounds,  Vol.  I.,  p  24. 


52  ORGANIC   ANALYSIS. 

at   1 80°.     It  melts  with  decomposition  to  a  red-brown  liquid  at 
i89°-i92°  (uncor.). 

DETERMINATION  BY  OXIDATION. 
lodimetric  Method.  * 

This  method  depends  upon  the  oxidation  of  formaldehyde  to 
formic  acid  by  means  of  iodine  in  alkaline  solution.  Two  atoms 
of  iodine  oxidize  one  molecule  of  formaldehyde  and  the  excess  of 
iodine  is  liberated  by  acidulation  and  determined  by  titration  with 
sodium  thiosulphate. 

Reagents.  —  Standard  solutions  of  iodine  and  sodium  thiosul- 
phate, preferably  about  tenth-normal.  Approximately  normal  so- 
lutions of  sodium  hydroxide  and  hydrochloric  acid. 

Determination.  —  Dilute  a  weighed  portion  of  the  sample  with  a 
known  quantity  of  water  so  as  to  obtain  a  solution  containing  0.5 
to  I  per  cent,  of  actual  formaldehyde.  Mix  10  c.c.  of  this  solution 
with  25  c.c.  normal  sodium  hydroxide  and  add  from  a  burette  50 
to  75  c.c.  of  tenth-normal  iodine  solution  or  enough  to  assure  an 
excess  of  iodine  as  shown  by  the  permanent  yellow  color  of  the 
solution.  Shake  or  stir  thoroughly  and  after  ten  minutes  add  35 
c.c.  normal  hydrochloric  acid  and  titrate  with  sodium  thiosulphate 
in  the  usual  way  using  starch  solution  as  indicator.  At  the  same 
time  determine  the  strength  of  the  iodine  in  terms  of  the  thiosul- 
phate solution  and  from  the  amount  of  iodine  consumed  in  oxidiz- 
ing the  formaldehyde  calculate  the  weight  of  the  latter  in  the  ten 
c.c.  taken  for  the  determination. 

Notes.  —  Under  the  conditions  given  the  oxidation  of  formalde- 
hyde is  rapid  and  complete  but  the  method  is  applicable  only  in 
the  absence  of  all  other  substances  capable  of  consuming  iodine 
under  these  conditions.  Other  aldehydes,  acetone  and  alcohol 
cause  high  results,  the  latter  probably  through  absorbing  iodine 
with  the  formation  of  chloroform. 

In  the  absence  of  interfering  compounds,  the  method  is  very 
satisfactory,  even  for  solutions  containing  only  one-tenth  per  cent, 
of  formaldehyde.  Variations  in  the  excess  of  iodine  added  have 
no  appreciable  influence  upon  the  results. 


*Romijn  :  Ztschr.  anal.  Chem.,  1897,  36,  18.     Williams  :  Essay  for  the  Degree  of 
Master  of  Arts,  Columbia  University,  November  1904. 


.      ALDEHYDES.  53 

Hydrogen  Peroxide  Method* 

In  this  method,  formaldehyde  is  oxidized  to  formic  acid  by  means 
of  hydrogen  peroxide  in  the  presence  of  a  known  amount  of  alkali. 
The  excess  of  alkali,  over  that  required  to  combine  with  the  formic 
acid  produced,  is  determined  by  titration. 

Reagents.  —  Standard  solutions  of  sodium  hydroxide  and  sul- 
phuric acid,  not  weaker  than  normal  nor  stronger  than  twice 
normal.  Neutral  f  3  per  cent,  solution  of  hydrogen  peroxide. 
Litmus  solution  as  indicator. 

Determination.  —  Weigh  2  to  3  grams  of  the  solution  to  be  tested 
in  a  stoppered  flask  or  bottle,  add  30  to  50  c.c.  normal  sodium 
hydroxide  and  then  add  cautiously  from  a  pipette  50  c.c.  of  the 
peroxide  solution,  insert  the  stopper  loosely  and  allow  to  stand  at 
room  temperature,  shaking  frequently  until  evolution  of  gas  ceases. 
Working  in  a  warm  room  with  double  normal  alkali  and  with 
samples  containing  over  30  per  cent,  of  formaldehyde  the  reaction 
takes  place  readily  with  considerable  production  of  heat,  and  is  com- 
plete within  ten  minutes.  If  normal  alkali  is  used  and  the  work 
is  done  in  a  cold  room  or  with  a  more  dilute  solution  of  formal- 
dehyde, an  hour  should  be  allowed  for  the  oxidation  of  the  formal- 
dehyde by  the  peroxide.  Finally  add  a  few  drops  of  litmus  solu- 
tion and  titrate  the  alkali  remaining  uncombined.  Each  molecule 
of  sodium  hydroxide  which  has  been  consumed  (deducting  the 
amount  required  to  neutralize  any  free  acid  which  the  peroxide 
solution  or  the  original  solution  of  formaldehyde  may  have  con- 
tained) represents  one  molecule  of  formaldehyde  oxidized  to 
formic  acid. 

Notes. — -For  the  best  results  the  strength  of  alkali  and  the  time 
allowed  for  the  reaction  should  be  governed  by  the  concentration 
of  the  formaldehyde  solution  tested.  Double  normal  alkali  is 
usually  recommended  and  the  reaction  is  said  to  be  complete  in 
ten  minutes  with  solutions  containing  as  little  as  5  per  cent,  of 
formaldehyde.  For  stronger  solutions  the  method  is  made  more 
accurate  by  using  normal  or  sesqui-normal  alkali  and  allowing  an 
hour  for  the  oxidation. 

Acetaldehyde  is  partially  oxidized  under  the  same  conditions. 
Its  presence,  therefore,  causes  high  results  but  not  so  high  as  by 


*  Blank  and  Finkenbeiner :   Ber.  deut.  chem.  Ges.,  1898,  31,  2979. 

f  If  all  available  peroxide  is  acid,  the  acidity  must  be  determined  by  titration,  using 
litmus  as  indicator,  and  allowed  for  in  calculating  the  amount  of  alkali  consumed  in 
the  formaldehyde  determination. 


54  ORGANIC   ANALYSIS. 

the  iodimetric  method.  The  results  are  not  influenced  by  the 
presence  of  paraldehyde,  acetone  or  ethyl  or  methyl  alcohol. 
Commercial  formalin  containing  only  traces  of  acetone  or  acetalde- 
hyde  should  show  the  same  percentage  of  formaldehyde  by  the 
peroxide  as  by  the  iodimetric  method. 

DETERMINATION  BY  CONDENSATION  REACTIONS. 

Several  of  the  condensation  reactions  of  formaldehyde  have  been 
utilized  for  its  quantitative  determination.  One  of  the  oldest  and 
best-known  methods  is  based  upon  the  fact  that  formaldehyde  and 
ammonia  when  mixed  in  not  too  dilute  solution  condense  to  form 
hexamethylene  tetramine : 

6CH20  +  4NH4OH  =  N4(CH2)6+  ioH2O. 

If  a  known  amount  of  ammonia  is  used,  the  determination  of  the 
excess  shows  the  amount  of  formaldehyde  originally  present. 

Legler's  Ammonia  Method* 

Weigh  about  1.5  grams  of  the  solution  containing  30  to  40  per 
cent,  formaldehyde,  or  an  equivalent  amount  of  a  more  dilute  solu- 
tion, into  a  250  c.c.  glass-stoppered  flask  or  bottle.  Add  100  c.c. 
of  fifth -normal  ammonia  solution;  stopper  tightly  at  once;  mix 
and  allow  to  stand  over  night  at  room  temperature.  Standing  for 
two  or  three  days  does  no  harm,  provided  the  stopper  fits  so 
tightly  as  to  prevent  any  loss  of  ammonia.  Finally,  add  a  very 
small  amount  of  rosalic  acid  as  indicator  and  titrate  the  excess  of 
ammonia  with  standard  sulphuric  acid.  Calculate  the  quantity  of 
formaldehyde  originally  present  from  the  amount  of  ammonia 
consumed  in  condensing  with  it  according  to  the  equation  given 
above. 

Notes  and  Precautions.  —  In  order  to  prevent  loss  of  ammonia 
during  the  determination,  the  flask  or  bottle  must  be  very  tightly 
closed,  the  stopper  being  coated  with  vaseline  if  necessary.  For 
the  same  reason  the  excess  of  ammonia  should  be  titrated  quickly 
after  opening  the  flask.  Normal  or  half-normal  ammonia  is  com- 
monly recommended  for  this  method,  but  the  fifth- normal  solution 
is  less  likely  to  lose  strength  and  has  been  found  by  Williams  to 
give  as  complete  reactions  as  the  stronger  solutions.  In  titrating 

*  Legler  :  Ber.  deut.  chem.  Ges.,  1883,  16,  1333.  Smith:  Journ.  Amer.  Chem. 
Soc.f  1903,  25,  1028.  Williams:  loc.  cit. 


ALDEHYDES.  55 

the  excess  of  ammonia  the  end  reaction  is  usually  unsatisfactory, 
especially  when  the  solution  is  highly  colored  by  the  indicator. 
Two  drops  of  a  freshly  prepared  o.i  per  cent,  solution  of  rosalic 
acid  has  been  found  sufficient.  With  a  view  to  obtaining  a  sharper 
end  reaction,  Williams  recommended  diluting  the  commercial 
formalin  so  as  to  obtain  a  solution  containing  about  2  per  cent,  of 
formaldehyde.  Mix  10  c.c.  of  this  solution  with  40  c.c.  of  fifth- 
normal  ammonia  in  a  small  glass-stoppered  bottle  and  complete 
the  determination  as  above  described.  The  method  is  thus  appli- 
cable to  solutions  containing  only  I  to  2  per  cent,  of  formaldehyde, 
although  its  results  in  such  cases  may  be  somewhat  low. 

The  results  are  not  affected  by  the  presence  of  acetone,  methyl 
or  ethyl  alcohol,  paraldehyde  or  benzaldehyde.  Acetaldehyde 
reacts  with  ammonia  and  thus  causes  high  results  if  present  in  the 
formaldehyde  solution. 

This  method,  carefully  carried  out  as  described,  gives  results 
lower  than  those  obtained  by  the  peroxide  method.  The  dis- 
crepancy varies  greatly  with  different  analysts  and  appears  to  be 
largely  dependent  upon  the  interpretation  of  the  end-point  in 
titrating  the  excess  of  ammonia.  The  color  change  is  not  sharp, 
but  by  using  very  little  indicator  a  fairly  definite  point  can  be  noted 
at  which  the  pink  tint  disappears,  leaving  the  solution  nearly  color- 
less. This  is  the  most  distinct  and  satisfactory  end-point.  It  has 
been  suggested,  however,  that  the  indistinctness  of  the  end  reaction 
may  be  due  to  the  faintly  basic  character  of  the  hexamethylene- 
tetramine,  and  that  the  first  indication  of  a  color  change  should  be 
taken  as  showing  the  point  at  which  the  excess  of  ammonia  is 
neutralized.  This  mode  of  titration  yields  higher  results  which 
often  agree  with  those  obtained  by  oxidation  methods.  As  yet 
there  is  no  standard  by  which  to  judge  whether  the  higher  or  the 
lower  results  are  more  nearly  correct. 

DETERMINATION  BY  ADDITION  REACTIONS. 

The  general  addition  reaction  of  aldehydes  with  bisulphites  has 
been  used  quantitatively  by  Ripper,  as  already  noted.  For  the 
determination  of  formaldehyde,  however,  the  reaction  with  potas- 
sium cyanide  has  been  found  especially  useful. 

Potassium  Cyanide  Method* 
On  mixing  aqueous  solutions  of  formaldehyde  and  potassium 

*  Romijn  :  Ztschr.  anal.  Chem.,  1897,  36,  1 8.     Smith  :  loc.  cit.     Williams  :  loc.  cit. 


56  ORGANIC  ANALYSIS. 

cyanide  an  addition  product  is  formed,  which,  according  to  Romijn, 
is  probably  the  potassium  compound  of  oxyacetonitril : 

CH2O  +  KCN  =  CH2OK-CN. 

The  addition  product  reduces  silver  nitrate  in  alkaline  solution, 
but  has  no  effect  in  the  presence  of  an  excess  of  nitric  acid.  If, 
therefore,  the  formaldehyde  to  be  tested  be  mixed  with  a  known 
solution  of  potassium  cyanide,  the  latter  being  in  excess,  and  the 
mixture  added  to  a  standard  solution  of  silver  nitrate  acidulated 
with  nitric  acid,  only  the  excess  of  potassium  cyanide  reacts  with 
the  silver  nitrate.  The  amount  of  formaldehyde  originally  present 
is  shown  by  the  quantity  of  potassium  cyanide  consumed  in  the 
formation  of  the  addition  product.  The  details  of  the  method  as 
here  given  are  nearly  identical  with  those  originally  recommended 
by  Romijn. 

Reagents.  —  Tenth-normal  solutions  of  silver  nitrate  and  ammo- 
nium thiocyanate.  A  solution  of  potassium  cyanide  6.2  grams  per 
liter.  Saturated  solution  of  ferric  ammonium  sulphate.  Nitric 
acid  1.32  sp.  gr.  (fifty  per  cent.). 

Determination. — (i)  Measure  15  c.c.  tenth-normal  silver  nitrate 
into  a  100  c.c.  flask,  add  6  to  8  drops  of  the  nitric  acid  and  10  c.c. 
of  the  cyanide  solution  ;  shake,  dilute  to  the  mark,  mix  thoroughly 
and  filter  through  a  dry  paper.  Titrate  50  c.c.  of  the  filtrate  with 
tenth-normal  ammonium  thiocyanate,  using  5  c.c.  of  the  ferric  so- 
lution as  indicator.  The  strength  of  the  silver  and  of  the  thiocya- 
nate solutions  being  known,  this  titration  shows  the  strength  of  the 
cyanide. 

(2)  Dilute  the  sample  until  it  contains  about  I  per  cent,  of  for- 
maldehyde, mix  10  c.c.  of  this  dilute  solution  with  35  c.c.  of  the 
cyanide  solution  and  rinse  the  mixture  into  another  portion  of 
1 5  c.c.  tenth-normal  silver  nitrate,  acidulated  with  6  to  8  drops  of 
the  nitric  acid  and  contained  in  a  100  c.c.  flask  ;  shake  and  deter- 
mine the  excess  of  silver  by  means  of  thiocyanate  in  the  same  way 
as  before.  Twice  the  difference  between  the  two  titrations  (since 
only  half  the  liquid  was  used  in  each  case)  represents  the  amount 
of  cyanide  consumed  by  the  formaldehyde.  If  the  thiocyanate 
solution  is  exactly  tenth-normal,  twice  the  difference  (in  c.c.)  be- 
tween the  two  titrations,  multiplied  by  0.003002,  gives  the  weight  of 
formaldehyde  (in  grams)  in  the  portion  taken  for  the  determina- 
tion. 


ALDEHYDES.  57 

Notes. — This  method  is  applicable  to  very  dilute  solutions  of 
formaldehyde,  larger  volumes  being  used  in  place  of  the  IO  c.c. 
called  for  in  the  above  directions.  Smith  obtained  accurate  results 
upon  a  solution  containing  o.oi  per  cent. 

Ethyl  and  methyl  alcohols,  acetone,  benzaldehyde  and  paralde- 
hyde  do  not  interfere.  Acetaldehyde  causes  high  results  if  allowed 
to  stand  for  some  time  in  contact  with  the  cyanide  solution,  but  if 
the  formaldehyde  solution  is  added  to  the  cyanide  and,  after  mix- 
ing, poured  at  once  into  the  silver  nitrate  solution,  the  presence  of 
acetaldehyde  does  not  influence  the  results.  With  commercially 
pure  solutions  of  formaldehyde  in  water  Romijn  and  Smith 
obtained  concordant  results  by  the  iodimetric  and  cyanide  meth- 
ods. Williams  obtained  concordant  results  by  the  ammonia  and 
the  cyanide  methods  which  were  lower  than  those  obtained  by  the 
oxidation  methods. 

ADDITIONAL  REFERENCES. 
Trillat:  Bull.  Soc.  Chim.,  1893,  [3],  9,  305. 
Brochet  and  Cambier:  Compt.  rentf-,  1895,  120,  449,  557. 
H.  M.  Smith  :  Analyst,  1896,  21,  148. 

Grlitzner:  Arch.  Pharm.,  1896,  234,  634;  Abs.  Ztschr.  anal. 
Chem.,  1897,36,  527. 

Orchard:  Analyst,  1897,  22,  4. 

C.  E.  Smith  :  Amer.Jour.  Pharm  ,  1898,  70,  86. 

Kebler:  Ibid.,  1898,  70,  432. 

Tollens  and  Clowes:  Ber.  deut.  chem.  Ges.,  1899,  32,  2841. 

Wolff:  Ztschr.  Unters.  Nahr.  Genussm.,  1900,  3,  87. 

Riegler:  Ztschr.  anal.  Chem.,  1901,  40,  92. 

Vanino  and  Seitter:  Ibid.,  1901,  40,  587. 

Vanino  :  Ibid.  1901,  40,  720. 

Craig:  Journ.  Amer.  Chem.  Soc.,  1901,  23,  638. 

Peska:  Chem.  Ztg.,  1901,  25,  743. 

Pfaff:  Ibid.,  1902,  26,  701. 

Scruff:  Ibid.,  1903,  27,  14. 

Lemme  :   Ibid.,  1903,  27,  896. 

Wallnitz:  Gerberztg.,  1903;   Abs.  Analyst,  1903,28,  189. 


CHAPTER  V. 

Carbohydrates  —  General  Methods. 

The  carbohydrates  include  the  simple  sugars  (monosaccharides) 
and  the  substances  which  can  be  converted  into  simple  sugars  by 
hydrolysis.  The  monosaccharides  are  aldehyde  alcohols  or  ketone 
alcohols,  each  molecule  containing  a  carbonyl  group  and  several 
hydroxyl  groups,  one  of  the  latter  being  adjacent  to  the  carbonyl 
group  (Meyer-Jacobson). 

The  purpose  of  this  chapter  is  to  outline  the  more  important 
analytical  properties  of  the  following  carbohydrates: 

Monosaccharides:  Hexoses  —  Dextrose  (d.  glucose),  Levulose 
(d.  fructose),  Galactose,  Mannose  ;  Pentoses  —  Xylose,  Arabinose. 

Disaccharides  :  Sucrose,  Lactose,  Maltose. 

Trisaccharide :  Raffinose. 

Polysaccharides :  Starch,  Dextrin,  Glycogen,  Galactan,  Cellulose, 
Pentosans. 

In  addition  to  a  few  notes,  on  the  occurrence,  relations  and  solu- 
bilities of  these  carbohydrates,  their  reactions  with  phenylhydrazine 
and  with  alkaline  copper  solutions,  their  behavior  on  treatment 
with  acids  and  their  power  of  rotating  the  plane  of  polarized  light 
will  be  considered. 

OCCURRENCE  AND  RELATIONS. 

Monosaccharides  (glucoses,  gly coses,  monoses)  have  the  com- 
position (CH2O)x*  and  are  called  tetroses,  pentoses,  hexoses,  etc., 
according  to  the  number  of  carbon  atoms  in  the  molecule.  Only 
pentoses  and  hexoses  are  of  sufficient  practical  importance  to  call 
for  consideration  in  connection  with  ordinary  methods  of  analysis. 
The  pentoses  do  not  occur  free  in  nature  but  are  met  by  the  analyst 
as  products  of  the  hydrolysis  of  the  pentosans.  The  hexoses  include 
all  of  the  monosaccharides  of  present  commercial  importance  and 
all  whose  biological  relations  have  been  thoroughly  studied. 

Dextrose  (d.  glucose,  grape  sugar,  starch  sugar,  diabetic  sugar, 
ordinary  glucose)  is  widely  distributed  in  nature,  occurring  especially 
in  fruits  and  plant  juices,  often  mixed  with  other  sugars.  It  is  a 

*  This  statement  does  not  apply  to  the  methyl  derivatives  now  frequently  classified 
as  monosaccharides. 

58 


CARBOHYDRATES— GENERAL  METHODS.        59 

normal  constituent  of  blood  and  is  the  form  of  carbohydrate 
ordinarily  found  in  the  urine  in  diabetes  or  glycosuria.  With  the 
exception  of  the  pentosans  and  galactan  all  of  the  di-,  tri-,  and 
polysaccharides  mentioned  above  yield  dextrose  on  hydrolysis. 

Levulose  (d.  fructose,  fruit  sugar)  occurs  with  dextrose  in  plant 
juices  and  especially  in  fruits  and  honey.  It  is  also  a  product  of 
the  hydrolysis  of  sucrose  and  of  raffinose. 

Galactose  does  not  occur  free  but,  as  a  product  of  hydrolysis  of 
lactose,  raffinose  and  the  galactans,  it  is  of  considerable  analytical 
importance. 

Mannose  also  is  not  found  free  but  has  been  detected  among  the 
products  of  hydrolysis  of  the  insoluble  carbohydrate  matter  of  a 
number  of  thick-walled  vegetable  tissues,  nut  shells,  etc.,  and  of 
several  Japanese  vegetables. 

The  disaccharides  considered  here  are  all  hexo-bioses  (C12H22On). 

Sucrose  (saccharose,  cane  sugar)  is  widely  distributed  in  the 
vegetable  kingdom  being  found  in  considerable  quantity,  generally 
mixed  with  dextrose  and  levulose,  in  the  fruits  and  juices  of  many 
plants.  The  most  important  sources  of  sucrose  are  the  sugar  beet, 
the  sugar  and  sorghum  canes,  and  the  sugar  maple.  A  molecule 
of  sucrose  yields  on  hydrolysis  one  molecule  each  of  dextrose  and 
levulose.  The  hydrolysis  of  sucrose  is  often  called  "  inversion  " 
and  the  resulting  mixture  of  equal  parts  dextrose  and  levulose  is 
known  as  "  invert-sugar." 

Lactose  (lactobiose,  milk  sugar)  occurs  in  the  milk  of  most  mam- 
mals, constituting  usually  from  4  to  7  per  cent,  of  the  fresh  secre- 
tion. Lactose  crystallizes  with  one  molecule  of  water  which  it 
retains  on  drying  at  room  temperature  over  sulphuric  acid  or  on 
heating  in  the  air  at  100°,  but  loses  at  about  130°.  A  molecule 
of  lactose  yields  on  hydrolysis  one  molecule  each  of  dextrose  and 
galactose. 

Maltose  (malt  sugar)  is  formed  from  starch  by  the  action  of 
diastatic  enzymes  and  is  therefore  an  important  constituent  of  ger- 
minating cereals,  malt,  malt  extract  and  beer  wort.  It  is  also 
formed  as  an  intermediate  product  when  starch  is  hydrolyzed  to 
dextrose  by  boiling  with  dilute  mineral  acids,  as  in  the  manufac- 
ture of  commercial  glucose.  Maltose  crystallizes  with  one  molecule 
of  water  which  it  loses  on  heating  in  the  air  at  100°.  Each  mole- 
cule of  maltose  yields  two  molecules  of  dextrose  on  hydrolysis. 

The   only   trisaccharide   of    practical    importance   is   raffinose 


6o  ORGANIC   ANALYSIS. 

(C]8H32O16)  also  called  meletriose  and  formerly  melitose  or  gossy- 
pose.  It  occurs  in  cotton  seed  and  in  small  quantity  in  the  germs 
of  various  other  seeds  including  wheat  and  barley.  Sugar  beets, 
especially  if  unhealthy  or  injured,  sometimes  contain  raffinose  in 
sufficient  quantity  to  affect  the  refining  process.  Raffinose  crys- 
tallizes with  five  molecules  of  water  in  needles  or  slender  prisms 
and  has  a  marked  influence  upon  the  crystallization  of  the  cane 
sugar  present.  *  Raffinose  loses  its  water  of  crystallization  at  1 00°. 
On  hydrolysis  it  yields  one  molecule  each  of  dextose,  levulose  and 
galactose.  Partial  hydrolysis  results  in  the  formation  of  levulose 
and  the  disaccharide,  melibiose. 

Starch  (C6H10O5)X  is  the  most  important  of  the  polysaccharides 
being  the  principal  form  of  carbohydrate  in  grains  and  most  other 
edible  seeds,  as  well  as  in  potatoes  and  other  tubers.  It  is  the  main 
product  of  the  assimilation  process  and  the  principal  reserve  car- 
bohydrate of  most  green  plants.  Commercially  it  is  of  great  im- 
portance as  a  constituent  of  foods,  as  the  source  of  dextrin,  maltose 
and  commercial  glucose  and  as  the  principal  raw  material  of  many 
of  the  fermentation  industries.  Starch  constitutes  over  one-half  of 
the  solid  matter  of  all  ordinary  cereals  and  about  three-fourths  of 
the  total  solids  in  potatoes.  Starch  granules  of  different  plants 
vary  in  size  and  structure  so  that  in  most  cases  the  source  of  a 
starch  which  has  not  been  altered  by  heat,  ferments,  or  chemical 
reagents,  can  be  determined  by  microscopical  examination.  All 
starches  yield  dextose  only,  as  the  final  product  of  complete 
hydrolysis. 

Dextrins,  (C6H10O5)x  or  (C6H10O5\-  H2O,  are  formed  from  starch 
by  the  action  of  enzymes,  acids  or  heat.  Small  amounts  of  dextrin 
are  found  in  normal,  and  larger  amounts  in  germinating,  cereals. 
Malt  diastase  acting  upon  starch  in  fairly  concentrated  solution 
yields  usually  about  one  part  of  dextrin  to  four  of  maltose.  During 
acid  hydrolysis,  dextrin  is  formed  as  an  intermediate  product 
between  soluble  starch  and  maltose.  Commercial  dextrin,  the 
principal  constituent  of  "  British  gum,"  is  obtained  by  heating 
starch,  either  alone  or  with  a  small  amount  of  dilute  acid. 

Glycogen,(C6H10O5)x  or  perhaps  (C6H10O.)x  •  H2O,  is  the  principal 
carbohydrate  of  the  animal  organism  being  found  in  small  quantity 
in  the  muscles  and  more  abundantly  in  the  liver  of  all  well- 
nourished  animals.  It  is  a  white  amorphous  powder  intermediate 

*  Stone  and  Baird :  Journ.  Amer.  Chem.  Soc.*  1897,  19,  116. 


CARBOHYDRATES— GENERAL  METHODS.        61 

in  properties  between  starch  and  dextrin  and  is  sometimes  called 
animal  starch.  The  determination  of  glycogen  is  often  important 
in  physiological  investigations  and  is  sometimes  useful  in  distin- 
guishing horseflesh  from  beef,  the  latter  containing  usually  less 
than  0.7  per  cent,  of  glycogen,  the  former  often  two  or  three  times 
this  amount.  On  complete  hydrolysis  glycogen  yields  only 
dextrose. 

Galactans,  amorphous  polysaccharides  yielding  galactose  on 
hydrolysis,  occur  in  small  quantity  in  many  plants  and  in  relatively 
large  amount  in  the  seeds  of  the  legumes  where  they  largely  replace 
starch  as  reserve  carbohydrate.  Since  the  galactans  are  readily 
hydrolyzed  by  hot  dilute  acids  and  are  digested  by  some  of  the 
diastatic  enzymes,  it  is  probable  that  galactan  has  been  reported  as 
starch  in  many  analyses. 

Cellulose  occurs  in  the  cell  walls  of  all  vegetable  tissues.  The 
term  is  sometimes  applied  to  the  whole  of  the  fiber  which  is  unat- 
tacked  by  boiling  dilute  acids  and  alkalies  but  should  be  restricted 
to  that  constituent  of  the  fiber  which  is  of  a  true  carbohydrate 
nature.  "Normal"  cellulose,  such  as  is  derived  from  cotton  and 
flax  fibers,  yields  dextrose  on  hydrolysis.  A  few  celluloses  have 
been  found  to  yield  mannose  or  a  pentose  (probably  xylose)  in 
addition  to  dextrose  (Tollens). 

Pentosans,  anhydrides  of  arabinose  and  xylose,  are  the  princi- 
cipal  constituents  of  the  vegetable  gums,  araban  occurring  especi- 
ally in  the  soluble  gums  such  as  cherry  gum  and  gum  arabic,  xylan 
in  the  so-called  woodgum  of  fibrous  tissues  such  as  wood,  straw, 
vegetables,  and  the  outer  portion  of  the  cereal  grains.  The  wheat 
grain,  for  example,  contains  3  to  5  per  cent,  of  pentosan  which  in 
the  milling  process  is  largely  left  in  the  bran.  The  so-called  patent 
flour  obtained  from  the  interior  of  the  grain  contains  hardly  any 
pentosan  while  the  breakfast  cereals  and  the  so-called  entire  wheat 
and  graham  flours  have  usually  about  as  much  as  the  original 
grain. 

SOLUBILITIES. 
IN  WATER. 

Of  the  carbohydrates  mentioned  above,  all  except  the  polysac- 
charides are  crystallizable  compounds  dissolving  in  water  to  form 
clear  solutions.  Milk  sugar  dissolves  in  six  parts  of  water  at  ordi- 
nary temperature;  all  of  the  other  members  are  more  freely  solu- 


62  ORGANIC   ANALYSIS. 

ble.  Among  the  polysaccharides,  dextrin,  glycogen  and  some 
of  the  galactans  and  pentosans  are  soluble  ;  starch,  cellulose,  some 
of  the  galactans  and  most  of  the  pentosans  of  ordinary  food  mate- 
rials are  insoluble  in  cold  water.  Glycogen  gives  a  strongly  opal- 
escent solution,  which  is  not  cleared  by  repeated  filtration  but 
loses  its  opalesence  on  the  addition  of  a  little  potassium  hydroxide 
or  acetic  acid.  On  heating  with  water,  starch  grains  swell  and  finally 
gelatinize  with  the  formation  of  "  starch  paste."  Different  starches 
vary  considerably  in  the  temperature  at  which  they  gelatinize  and 
in  the  physical  properties  of  the  paste  produced.  Thin  starch  pastes 
can  be  filtered  through  paper,  but  almost  always  leave  some  gela- 
tinous residue  upon  the  filter.  Pastes  containing  only  a  few  hun- 
dredths  of  one  per  cent  of  starch  become  clear  on  boiling  and  can 
be  filtered  without  loss.  Water-soluble  starch  can  be  prepared  * 
by  chemical  treatment  and  is  sometimes  found  in  natural  products, 
for  example  in  immature  grains. 

Cellulose,  and  the  ordinary  pentosans  of  foods  and  fibers,  are 
insoluble  in  water  and  not  gelatinized  by  boiling. 

IN  ALCOHOL  AND  ETHER. 

Levulose  is  soluble  in  5  parts  of  cold  absolute  alcohol  and  is 
somewhat  soluble  in  mixtures  of  ether  and  strong  alcohol.  The 
other  monosaccharides  are  sparingly  soluble  in  cold  alcohol,  in- 
soluble in  ether  and  practically  insoluble  in  the  alcohol-ether 
mixture.  Dextrose  is  much  more  readily  soluble  in  hot  than  in 
cold  alcohol ;  100  parts  of  90  per  cent,  alcohol,  dissolve  about  2 
parts  dextrose  at  1 8°,  about  22  parts  at  boiling  temperature. 

The  di-,  tri-,  and  polysaccharides  are  insoluble  in  ether.  Di- 
and  trisaccharides  are  less  soluble  in  alcohol  than  is  dextrose. 
Lactose  is  practically  insoluble  in  alcohol,  even  when  the  latter  is 
diluted  to  60  per  cent. 

Sucrose  is  much  more  readily  soluble  in  diluted  than  in  concen- 
trated alcohol.  According  to  Scheibler : 

loo  parts  90  per  cent,  alcohol  dissolve    0.9  parts  at  14°  ;      2.3  parts  at  40°. 

«.        «       go     «         tt  it  «  6.6       «        u       a         j.j  3       «       «       « 

«      «     70    «       «          «  «       jg  g     «     «     «      3!  4     «     «     « 

«      «     60    «      •'          "  "       33.9     "     "     "      49-9     "     "     " 

"      «     50    "      "          "  "       47.1     "     "     "      63.4     "     "     " 

*Lintner:  Journ.  prakt.  Chem.  1886,  [2]  34,  381.  Wroblewski  :  Ztschr.  physiol. 
Chem.,  1898,  24,  173.  Vaubel :  Best.  org.  Verbind.,  II.,  500. 


CARBOHYDRATES— GENERAL  METHODS.        63 

Sucrose  dissolves  in  about  80  parts  of  boiling  absolute  alcohol. 

All  of  the  polysaccharides  are  insoluble  in  alcohol.  Those 
which  are  soluble  in  water  can  be  precipitated  from  their  aqueous 
solutions  by  the  addition  of  strong  alcohol. 

IN  ACIDS  AND  ALKALIES. 

Among  the  carbohydrates  which  are  inslouble  in  water,  separa- 
tions can  sometimes  be  made  by  the  use  of  acid  or  alkaline  solu- 
tions. 

Cellulose  is  soluble  in  concentrated  sulphuric  acid,  but  insoluble 
in  any  ordinary  aqueous  solution  of  acid  or  alkali.  It  dissolves  in 
Schweitzer's  reagent  (aqueous  ammonia  saturated  with  cupric  hy- 
droxide) to  a  viscous  solution  from  which  it  is  precipitated  by 
neutralization  with  acid. 

Starch  is  insoluble  in  Schweitzer's  reagent  or  in  solutions  of  am- 
monia. Treated  with  dilute  aqueous  solutions  of  sodium  or  potas- 
sium hydroxide,  starch  swells,  gelatinizes  and  becomes  soluble.  It 
can  be  completely  precipitated  from  such  a  solution  by  neutralizing 
with  acetic  acid  and  adding  strong  alcohol.  Starch  is  not  affected 
by  dilute  solutions  of  alkalies  in  strong  alcohol.  By  diluted  solu- 
tions of  strong  acids  starch  is  first  dissolved,  then  •  hydrolized. 
Some  weak  organic  acids  dissolve  starch  with  little  if  any  hydro- 
lysis. Boiling  water  containing  I  per  cent,  of  salicylic  acid  dis- 
solves starch  to  an  opalescent  solution  which  filters  much  more 
readily  than  a  corresponding  starch  paste  made  with  water  alone. 

The  pentosans  of  foods  and  fibers  —  so-called  woodgums  con- 
sisting mainly  of  xylan  —  are  insoluble  in  dilute  ammonia  or  in 
Schweitzer's  reagent,  largely  soluble  in  dilute  aqueous  solutions  of 
sodium  or  potassium  hydroxide  and  in  cold  dilute  acids.  From 
such  solutions  the  pentosan  is  precipitated  by  alcohol.  Boiling 
dilute  mineral  acids  dissolve  and  hydrolize  pentosans  almost  as 
readily  as  starch.  Hence  pentosans  have  frequently  been  reported 
as  starch  when  the  latter  has  been  estimated  by  direct  hydrolysis 
with  acid  and  determination  of  the  resulting  glucose. 

f 

REACTIONS   WITH    PHENYLHYDRAZINE. 

All  of  the  monosaccharides,  and  maltose  and  lactose  among  the 

disaccharides,  contain  the  free  carbonyl  radical  and,  therefore,  on 

treatment  with  phenylhydrazine  acetate  in  molecular  proportions, 

they  react  to  form  hydrazones.     All  of  these  hydrazones,  except 


64  ORGANIC   ANALYSIS. 

that  of  mannose,  are  freely  soluble  in  water.  The  formation  of  an 
insoluble  hydrazone  is  a  distinctive  test  for  mannose,  and  has  been 
applied  as  a  quantitative  method  by  Bourquelot  and  Herissey.* 

When  the  hydrazone  of  a  sugar  is  heated  with  an  excess  of 
phenylhydrazine,  the  hydroxyl  adjacent  to  the  hydrazone  group 
undergoes  oxidation  and  condensation  resulting  in  the  formation 
of  the  dihydrazone,  more  commonly  known  as  the  osazone.  The 
osazones  are  only  sparingly  soluble  in  water,  and  on  crystallization 
show  definite  forms  and  melting  points.  The  formation  of  the  osa- 
zone is,  therefore,  an  important  means  of  identification  for  sugars 
having  the  carbonyl  radical. 

PREPARATION  AND  PROPERTIES  OF  THE  OSAZONES. 

loform  the  osazone,  f  dissolve  one  part  of  the  sugar,  two  of  pure 
phenylhydrazine  hydrochloride  and  three  of  crystallized  sodium 
acetate  in  20  parts  of  water  in  a  test-tube.  Cork  loosely  to  avoid 
evaporation  and  place  the  tube  in  boiling  water. 

If  the  substance  tested  is  pure  and  the  above  proportions  are 
maintained,  the  time  required  for  the  appearance  of  an  osazone 
precipitate  is  fairly  constant.  These  differences  in  the  rate  of 
osazone  formation  have  been  especially  studied  by  Mulliken  and 
play  a  prominent  part  in  his  system  of  identification  of  pure 
sugars.  All  of  the  monosaccharides,  and  no  other  carbohy- 
drates, give  precipitates  within  20  minutes.  Maltose  and  lactose 
form  osazones  which  are  much  more  soluble  in  hot  water  than 
those  of  the  monosaccharides,  and  under  the  conditions  given, 
separate  from  the  solution  only  on  cooling.  Sucrose  when  sub- 
jected to  this  test  is  gradually  hydrolyzed  so  that  eventually  it 
yields  the  osazone  of  dextrose  and  levulose.  Under  the  conditions 
given,  however,  this  requires  about  30  minutes  heating  in  the 
water  bath. 

In  general,  therefore,  a  precipitate  appearing  at  the  temperature 
of  the  water  bath  within  20  minutes  indicates  the  presence  of  a 
monosaccharide ;  a  slight  precipitate  of  osazone  appearing  only 
on  cooling  might  be  due  to  a  very  small  amount  of  monosacchar- 
ide, but  any  considerable  quantity  of  osazone  which  precipitates 

*  Journ.  Pkarm.  Chim.,  1899,  [6j,  10,  206.  See  also  Pellet :  Bull.  Assoc.  Chirn. 
Sucr.  Distill.,  1900-01,  18,  758;  Abs.  Zlschr.  Unters.  Nahr.  Gfnussm.,  1902,  5,  74. 

t  Fischer:  Ber.  deut.  chem.  Ges.,  1884,  17,  579;  1887,  20,  821  ;  1888,  21, 
1805,  2631  ;  1889,  22,  87  ;  1890,  23,  2117. 

Mulliken  :  Identification  of  Pure  Organic  Compounds,  Vol.  I.,  p.  32. 


CARBOHYDRATES— GENERAL  METHODS.        65 

only  on  cooling  is  indicative  of  lactose,  maltose   or  isomaltose. 

The  maximum  yield  of  osazone  is  usually  obtained  by  warming 
the  solution  in  the  water-bath  for  one  to  two  hours  and  then  allowing 
it  to  cool.  The  osazone  thus  obtained  is  ordinarily  a  yellow  iri- 
descent precipitate,  more  or  less  distinctly  crystalline  according  to 
the  purity  and  concentration  of  the  solution.  To  purify  it,  filter 
on  a  small  paper,  wash  with  a  little  cold  water,  dissolve  in  the 
smallest  possible  amount  of  boiling  50  per  cent,  alcohol  and  filter 
hot.  The  osazone  which  separates  from  the  alcohol  solution  may, 
if  desired,  be  further  recrystallized  from  alcohol  or  from  pyridine. 

Dextose,  levulose  and  mannose  yield  the  same  osazone,  glucosa- 
zonc,  which  crystallizes  in  needles  melting  at  2O4°-2O5°  when 
heated  at  such  a  rate  that  the  melting-point  is  reached  in 
three  to  four  minutes.*  Galactosazone  crystallizes  in  needles  which 
melt  at  193°;  maltosazone,  in  independent  needles  or  tables  melting 
at  206°;  lactosazone \  in  masses  of  microscopic  prisms  melting  at 
200°.  Xylosazone  and  arabinosazone  melt  at  about  1 60°. 

A  conclusive  method  of  ascertaining  whether  an  osazone  is  that 
of  a  pentose,  a  hexose  or  a  dtsaccharide  is  to  determine  the  per- 
centage of  nitrogen.  In  the  case  of  a  mixture,  the  hexosazone 
can  be  freed  from  osazones  of  the  other  two  groups  by  washing 
with  hot  water.  Maltosazone  is  soluble  in  about  75  parts,  xylosa- 
zone  in  about  50  parts,  and. lactosazone  in  80  to  90  parts  of  boiling 
water,  while  the  pure  hexosazones  are  nearly  insoluble. 

T/te  yield  of  osazone  has  been  studied  especially  by  Macquenne,f 
Laves,  +  Fischer,  §  Lintner  and  Krbber,  ||  Raimann,^f  and  Davis 
and  Ling.**  In  each  of  these  cases  the  osazone  was  formed  by 
heating  for  one  or  two  hours,  in  a  more  dilute  solution  than  that 
above  given,  with  a  considerable  excess  of  a  slightly  acid  solution 
of  phenylhydrazine  acetate.  The  precipitate  of  osazone  can  then 
be  filtered,  washed  with  water  and  weighed.  According  to  Laves 

*  Any  of  the  osazones  on  being  heated  very  slowly  may  begin  to  melt  considerably 
below  the  temperatures  given. 

•\Compt.  rend.,  1891,  na,  799;   Ztschr.  anal.  Chem.,  1894,  33,  226. 

\Archiv.  der  Pharm.,  1893,  231,  366;  Vaubel's  Bestimmung  organischer  Ver- 
bindungen,  II.,  307,  311,  312. 

\  Ber.  deut.  chem.  Ges.,  1895,  28,  1437. 

||  Ztschr.  f.  d.  ges.  Brauwesen,  1895.  18,  153  ;  Ztschr.  anal.  Chem.,  1896,  35,  95  ; 
Analyst,  1897,  20,  167. 

fl  Ztschr.  anal.  Chem.,  1901,  40,  390. 

**  Journ.  Chem.  Soc.,  1904,  85,  24. 


66  ORGANIC   ANALYSIS. 

the  following  corrections  should  be  applied  for  the  amount  of  glu- 
cosazone  left  in  solution  or  dissolved  by  washing : 

100  parts  boiling  water  dissolve  o.oi     part  osazone. 

100  "     water  at  20°        "  0.0042  "  " 

100  "     2  per  cent,  acetic  acid  at  20°  dissolve  0.067  "  " 

100  "     3    "      "        "        "     "  20°       "  0.0145  "  " 

100  "     4    "      "        "        "     "  20°       "  0.022  "  " 

100  "     5    "      "       "        "     "  20°       "  0.031  "  " 

ioo  "  10    "       "     alcohol  slightly  acidulated  dissolve  at  20°      0.0075  "  " 

Corresponding  corrections  have  not  been  worked  out  for  other 
osazones,  as  the  attempts  to  apply  the  osazone  method  quantita- 
tively have  been  mainly  with  the  object  of  determining  dextrose 
or  dextrose  and  levulose.  In  comparative  tests  carried  out  in  ex- 
actly the  same  manner,  the  weight  of  osazone  obtained  is  propor- 
tional to  the  amount  of  sugar  originally  present,  but  slight  differ- 
ences of  manipulation  affect  the  yield  to  such  an  extent  that  the 
osazone  precipitation  cannot  yet  be  regarded  as  a  satisfactory 
quantitative  method. 

For  the  detection  of  dextrose,  or  invert  sugar,  in  the  absence  of 
other  monosaccharides,  the  osazone  test  may  be  applied  as  follows :  * 
Dissolve  0.2  gram  of  sample  (or,  if  only  partially  soluble,  stir  so 
much  as  contains  0.2  gram  of  soluble  solids)  in  4  c.c.  of  water, 
filter  if  necessary,  add  0.40  gram  phenylhydrazine  hydrochloride 
and  0.60  gram  crystallized  sodium  acetate;  shake  until  dissolved, 
filter  if  not  clear  ;  place  the  solution,  contained  in  a  small  test-tube 
corked  loosely  to  prevent  evaporation,  in  a  beaker  of  boiling  water 
and  shake  frequently  without  removing  the  tube  from  the  water- 
bath. 

The  following  results  were  obtained  with  solutions  containing 
known  amounts  of  pure  sugars,  the  amounts  of  water  and  reagents 
and  the  manipulation  being  in  each  case  as  just  described. 

SOLUTIONS  OF  PURE  DEXTROSE. 

Dextrose  present  o.io         0.05         0.02         o.oi         0.005  gram. 

Osazone  appeared  in  5  6^          IO^£        I7~I9         34      minutes. 

SOLUTIONS    CONTAINING    o.oi   GRAM  DEXTROSE,  WITH  VARYING  AMOUNTS    OF 

MALTOSE  OR  LACTOSE. 

Maltose  or  Lactose  present         o.oi  0.05  o.io  0.20     gram 

Osazone  appeared  in  22-25        3°~32        37-4-6  t       minutes. 

*  Compare  Mulliken,  loc,  cit. 

t  No  precipitate  of  osazone  appeared  in  the  hot  solution  although  the  heating  was 
continued  for  two  hours. 


CARBOHYDRATES— GEXERAL  METHODS.        67 

These  experiments,  made  by  R.  H.  Williams  in  the  course  of  an 
investigation  which  is  still  in  progress  in  this  laboratory,  illustrate 
the  delicacy  of  the  test  when  only  dextrose  is  present,  and  the  influ- 
ence of  maltose  and  lactose  in  retarding  the  precipitation  of  dex- 
trose as  osazone.  Sucrose  up  to  ten  times  the  weight  of  dextrose 
present  had  no  appreciable  influence.  In  larger  proportion  it  may 
hasten  very  slightly  the  appearance  of  the  osazone.  As  already 
explained  sucrose  tested  alone  undergoes  gradual  hydrolysis  and 
thus  finally  gives  a  precipitate  of  glucosazone. 

The  delicacy  of  the  test  in  the  presence  of  dextrin  (which  is 
known  to  retard  the  formation  of  the  osazone),  and  of  substances 
other  than  carbohydrates,  has  not  yet  been  studied  in  detail,  except 
in  the  case  of  certain  constituents  of  urine.*  Hence  in  applying 
the  osazone  test  to  an  unknown  solution,  it  is  well  to  make  at  the 
same  time  two  check  experiments,  one  with  a  mixture  of  carbo- 
hydrates corresponding  to  that  which  the  unknown  solution  is 
believed  to  contain,  the  other  with  a  portion  of  the  unknown  solu- 
tion to  which  has  been  added  a  very  small  amount  of  dextrose. 

REDUCTION   OF   ALKALINE  COPPER   SOLUTIONS. 

The  aldehyde  and  ketone  sugars  are  easily  oxidized  and  hence 
have  the  power  of  reducing  certain  metallic  salts,  especially  salts  of 
copper,  silver  and  mercury  in  alkaline  solution.  The  reagent  most 
commonly  used  in  detecting  or  determining  sugars  by  their  "  reduc- 
ing power,"  is  prepared  by  treating  cupric  sulphate  with  an  excess 
of  an  alkaline  tartrate  solution  When  such  an  alkaline  copper 
solution  is  boiled  with  any  one  of  the  aldehyde  or  ketone  sugars 
(i.  e.,  with  any  carbohydrate  capable  of  reacting  with  phenylhydra- 
zine),  the  copper  is  reduced  by  the  action  of  the  sugar  from  the 
cupric  to  the  cuprous  state.  In  the  case  of  a  tartrate  solution  con- 
taining only  fixed  alkali,  the  reduced  copper  is  precipitated  as 
cuprous  oxide.  The  changes  which  take  place  in  the  sugar  are 
quite  complex  and  only  in  the  case  of  dextrose  have  the  products 
been  studied  to  any  extent. 

On  boiling  with  the  alkaline  copper  solution,  dextrose  is  attacked 
in  two  ways  :  f  (i)  Oxidation  to  tartronic,  gluconic,  oxalic,  acetic, 

*  The  influence  of  other  substances  likely  to  be  present,  upon  the  osazone  test  for 
dextrose  in  urine  has  been  studied  by — Hirschl ;  Ztschr.  physiol.  Chem.,  1890,  14, 
377  :  Jaffe  ;  Ibid.,  22,  532  :  Neuberg  ;  Ibid.,  1900,  29,  274  :  Neuraan-Wender  ;  Phar- 
mac.  Post.,  26,  573,  614  (Vaubel ;  /.  c.  II.,  309). 

t  For  fuller  discussions  of  this  subject  see  the  works  of  Lippmann,  Vaubel  and  Wiley 
cited  below. 


68  ORGANIC   ANALYSIS. 

formic  and  carbonic  acids,  and  probably  other  products  which  have 
not  yet  been  recognized.  (2)  Decomposition  by  alkali  into  sub- 
stances no  more  highly  oxidized  than  the  original  sugar.  Lactic 
acid  is  the  most  conspicuous  of  these,  but  other  more  complex 
products,  including  aromatic  compounds  and  gummy  substances  of 
unknown  constitution,  are  formed  by  rearrangement  under  the  in- 
fluence of  the  alkali.  In  practice  less  than  half  as  much  copper  is 
reduced  as  would  correspond  to  a  complete  oxidation  of  dextrose 
to  tartronic  acid,  although  the  latter  compound  is  probably  the 
chief  oxidation  product  formed.  It  is  evident,  therefore,  that  the 
decomposing  action  of  the  alkali,  unless  carefully  controlled,  will 
influence  greatly  the  amount  of  copper  reduced. 

Fehling,*  who  first  systematically  studied  the  determination  of 
reducing  sugars  by  their  action  upon  alkaline  copper  solutions, 
used  a  reagent  containing  34.64  grams  of  crystallized  copper  sul- 
phate, 138.6  grams  of  potassium  tartrate  and  47  to  55  grams  of 
sodium  hydroxide,  the  whole  dissolved  in  water  and  diluted  to  one 
liter.  Ten  c.c.  of  this  reagent  were  diluted  with  40  c.c.  of  water  and 
heated  to  boiling.  A  solution  containing  not  more  than  I  per  cent, 
of  dextrose  or  invert-sugar  was  then  added,  until  after  two  minutes 
boiling  the  solution  contained  no  unreduced  copper.  Fehling  held 
that  under  these  conditions  each  molecule  of  dextrose  or  levulose 
reduced  five  atoms  of  copper  from  the  cupric  to  the  cuprous  state. 
According  to  this  proportion  10  c.c.  of  the  Fehling  reagent  require 
for  complete  reduction  exactly  0.05  grams  of  dextrose,  levulose  or 
invert  sugar. 

It  has  been  found,  however,  that  the  ratio  between  dextrose  and 
copper  is  constant  only  when  the  alkalinity  and  dilution  of  the 
solution  are  kept  nearly  uniform.  According  to  Soxhlet,|  one 
molecule  of  dextrose  in  I  per  cent,  solution  reduces  5.26  atoms  of 
copper  from  undiluted  Fehling  solution  or  5.055  atoms  from  the 
same  solution  diluted  with  four  times  its  volume  of  water.  Soxhlet 
recommended  the  use  of  I  per  cent,  solutions  of  reducing  sugars 
with  undiluted  Fehling  solution  under  which  conditions  10  c.c.  of 
the  latter  are  reduced  by  0.0475  gram  of  dextrose.  The  use  of 
undiluted  Fehling  solution  has,  however,  the  disadvantage  that 
sucrose,  if  present,  may  be  partially  hydrolyzed  by  the  boiling 
alkali  thus  causing  high  results,  whereas  the  diluted  Fehling  solu- 

*  Ann.  Chem.,  1849,  72,   106. 

^Journ.  prakt.  Chetn.,  1880  [2],  21,  227. 


CARBOHYDRATES— GENERAL  METHODS.        69 

tion  seems  to  be  entirely  without  effect  upon  sucrose.  More- 
over, it  is  evident  from  the  figures  given  by  Soxhlet  that  at  a 
slightly  greater  dilution,  as  when  a  solution  containing  0.25  to  0.5 
per  cent,  of  dextrose  is  used,  the  ratio  of  Fehling  is  practically  cor- 
rect. Soxhlet  also  showed  that  the  reducing  power  is  constant 
only  when  the  whole  amount  of  sugar  required  for  the  reduction  of 
the  copper  is  added  at  once. 

Many  other  modifications  of  Fehling's  method  have  been  pro- 
posed and  a  committee  of  the  International  Commission  for  Uni- 
fying Methods  of  Sugar  Analysis  is  now  (1905)  investigating  this 
subject  with  a  view  to  the  development  of  more  satisfactory  and 
uniform  methods.  Until  some  such  international  agreement  is 
reached  the  volumetric  determination  of  reducing  sugars  may  con- 
veniently be  made  by  the  following  method,  which  is  that  of  Fehling 
with  only  such  modifications  as  have  been  generally  recognized  as 
essential  to  accuracy. 

FEHLING'S  VOLUMETRIC  METHOD. 

Reagents.  —  (i)  Copper  solution  :  Dissolve  34.64  grams  of  pure 
crystallized  copper  sulphate  in  water,  and  dilute  to  exactly  500  c.c. 

(2)  Alkaline  tartrate  solution :  Dissolve  175  grams  of  pure 
sodium  potassium  tartrate  and  50  grams  of  pure  sodium  hydroxide 
in  water,  and  dilute  to  500  c.c.  This  reagent  does  not  keep  well 
unless  carefully  protected  from  the  air. 

Determination.  —  Measure  accurately  into  a  small  flask  or  casserole 
or  a  deep  porcelain  dish,  5  c.c.  of  each  of  the  above  solutions, 
making  10  c.c.  of  the  "  mixed  Fehling  reagent."  Add  40  c.c.  of 
water,  mix  and  boil.  To  the  boiling  liquid,  add  from  a  burette  a 
solution  which  is  judged  to  contain  about  I  per  cent,  of  the  sugar 
to  be  determined,  until  after  two  minutes  boiling  the  blue  color  is 
entirely  discharged  showing  that  all  of  the  copper  has  been  reduced. 
This  test  indicates  approximately  the  amount  of  reducing  sugar  in 
the  sample.  Now  adjust  the  strength  of  the  sugar  solution,  if  neces- 
sary, so  that  between  10  and  20  c.c.  will  be  required  to  reduce  10 
c.c.  of  the  mixed  Fehling  reagent.  Repeat  the  test,  adding  the  cal- 
culated amount  of  sugar  solution  at  once  to  the  boiling  copper  solu- 
tion; regulate  the  heating  so  that  the  mixture  will  begin  to  boil 
about  one  minute  after  the  addition  of  the  sugar ;  note  the  exact 
time  that  actual  boiling  commences  and  continue  to  boil  for  just  two 
minutes,  then  remove  the  flame  and  at  once  test  the  liquid  for  un- 


70  ORGANIC   ANALYSIS. 

reduced  copper.  This  may  be  done  in  several  ways.  The  method 
commonly  recommended  is  to  filter  a  portion  of  the  liquid,  acidulate 
with  acetic  acid  and  test  for  copper  with  potassium  ferrocyanide. 
A  more  convenient  method  which  appears  to  be  equally  reliable 
has  recently  been  suggested  by  Harrison  *  and  consists  in  adding 
a  drop  or  two  of  the  liquid  (which  need  not  be  filtered  from  cuprous 
oxide)  to  a  considerable  excess  of  cold  acidulated  starch-iodide  so- 
lution prepared  as  described  below.  Any  unreduced  copper  causes 
a  liberation  of  iodine  with  the  production  of  a  color  varying  from 
purplish  red  to  deep  blue  according  to  the  amount  of  copper  in  the 
solution.  Until  considerable  experience  has  been  acquired,  it  is 
well  to  make  both  tests,  observing  all  the  precautions  noted  below. 

Repeat  the  test  using  more  or  less  of  the  sugar  solution  depend- 
ing upon  the  presence  or  absence  of  an  excess  of  copper  in  the 
preceding  experiment  until  two  amounts  of  sugar  solution  are  found 
which  differ  by  only  O.I  or  0.2  c.c.,  one  giving  complete  reduction 
and  the  other  leaving  a  small  amount  of  copper  in  the  solution. 
The  mean  of  the  two  readings  is  taken  as  the  volume  of  solution 
required  for  complete  reduction  of  the  copper  reagent. 

Notes  and  Precautions.  —  All  of  the  reagents  used  must  be  the 
purest  obtainable  and  the  two  constituents  of  the  "  mixed  Fehling 
reagent  "  must  be  kept  separate  instead  of  being  made  up  in  one 
solution  as  was  formerly  done.  If  the  solutions  are  not  fresh, 
make  a  blank  test  in  a  casserole,  boiling  as  above  without  the  addi- 
tion of  any  sugar,  allow  to  stand  for  a  few  minutes,  then  decant 
off  the  liquid  and  notice  whether  any  cuprous  oxide  has  been 
precipitated.  Finally  wipe  out  the  casserole  with  a  small  piece  of 
filter  paper  and  examine  the  latter,  which  may  show  traces  of 
cuprous  oxide  not  visible  in  the  presence  of  the  blue  solution.  If 
the  liquid  shows  any  change  of  color  in  this  blank  test,  or  if  the 
slightest  trace  of  cuprous  oxide  is  found,  the  alkaline  tartrate  solu- 
tion must  be  rejected  and  another  blank  test  made  with  a  freshly 
prepared  solution. 

If  the  sample  analyzed  is  rich  in  reducing  sugar  all  of  the  quan- 
tities given  above  should  be  doubled  in  order  to  reduce  the  influ- 
ence of  unavoidable  errors. 

In  testing  for  copper  by  the  ferrocyanide  method,  great  care 
must  be  taken  to  filter  out  all  of  the  cuprous  oxide  quickly.  This 
is  best  done  by  withdrawing  the  portion  to  be  tested  through  the 

*  Pharm.  Journ.,  1903,  170;  Button's  Volumetric  Analysis  (gthEd. ),  p.  312. 


CARBOHYDRATES— GENERAL  METHODS.        71 

filtering  tube  described  by  Wiley  *  or  by  pouring  it  carefully  upon 
a  double  or  triple  paper  filter.  A  single  filtration  through  one  or- 
dinary paper  seldom  removes  all  of  the  cuprous  oxide.  On  the 
other  hand  if  a  slight  trace  of  unreduced  copper  is  present  this  may 
sometimes  be  lost  by  repeatedly  passing  the  solution  through  sev- 
eral thicknesses  of  paper.  Traces  of  iron  getting  into  the  solution 
from  reagents  or  apparatus  may  cause  a  slight  coloration  with  fer- 
rocyanide.  Only  a  distinct  red-brown  precipitate  or  coloration 
should  be  taken  as  showing  the  presence  of  unreduced  copper. 

The  starch-iodide  solution  for  detecting  unreduced  copper  with- 
out filtration  is  prepared  as  follows :  Boil  0.02  gram  starch  with 
15  to  20  c.c.  of  water,  cool,  add  4  to  5  grams  potassium  iodide  and 
dilute  to  50  c  c.  A  fresh  solution  must  be  prepared  each  day. 
When  the  test  is  to  be  made  pour  about  I  c.c.  of  the  starch- iodide 
into  a  test-tube,  add  two  or  three  drops  of  acetic  acid  and  then 
immediately  a  drop  or  two  of  the  solution  to  be  tested. 

Calculation  and  Verification  of  Results. —  In  calculating  the  results 
it  is  commonly  assumed  that  10  c.c.  of  the  mixed  Fehling  reagent 
require  for  reduction  under  the  above  conditions  : 

0.0500  gram  of  anhydrous  dextose,  levulose  or  invert  sugar. 
0.0678     "       "  dry  crystallized  lactose  (C12HMOU  •  H2Oj. 
0.0807     "       "  anhydrous  maltose. 

That  these  factors  are  not  absolute,  but  are  dependent  upon  ex- 
act uniformity  of  conditions  has  already  been  explained.  To  secure 
the  greatest  accuracy,  therefore,  the  result  obtained  should  be  veri- 
fied by  a  check  experiment,  carried  out  under  the  exact  conditions 
of  the  analysis,  with  a  known  solution  of  pure  sugar  of  the  kind 
actually  determined. 

According  to  Soxhlet,  levulose  and  galactose  have  distinctly  less 
reducing  power  than  dextrose.  It  is  quite  commonly  assumed,  how- 
ever, that  these  three  monosaccharides  have  the  same  reducing 
power,  but  that  invert  sugar  often  fails  to  show  its  full  effect  because 
of  the  decomposing  action  of  the  acid  used  for  inversion.  Xylose 
and  arabinose  reduce  Fehling  solution  somewhat  more  strongly 
than  does  dextrose. 

Substitution  of  Other  Methods. 

Aside  from  modifications  in  detail,  several  methods  intended  to 
replace  that  of  Fehling  have  been  proposed.  In  some  of  these  the 

*  Agricultural  Analysis,  Vol.  III.,  p.  130. 


72  ORGANIC   ANALYSIS. 

caustic  alkali  of  Fehling's  solution  is  replaced  by  a  weaker  base, 
such  as  alkaline  carbonate  or  bicarbonate  (Soldani,  Sidersky,  Ost) 
or  ammonia  (Allein  and  Gaud);  in  others,  ammonia  (Pavy)  or 
cyanide  (Gerrard)  is  added  to  the  usual  Fehling  reagent  in  order 
to  hold  in  solution  the  reduced  copper,  so  that  the  point  of  com- 
plete reduction  can  be  directly  observed  by  the  disappearance  of 
the  blue  color.  More  important,  however,  are  the  methods  in 
which,  as  described  below,  a  fixed  volume  of  the  sugar  solution  is 
boiled  with  an  excess  of  the  alkaline  copper  solution  and  the  re- 
duced copper  collected  and  determined. 

DEFREN'S  GRAVIMETRIC  METHOD.* 

This  method  is  based  on  that  of  O'Sullivan  f  and  provides  a  uni- 
form procedure  for  the  determination  of  dextrose,  maltose  or 
lactose. 

Reagents.  —  (i)  Dissolve  34.64  grams  of  copper  sulphate  in 
water,  add  0.5  c.c.  strong  sulphuric  acid  and  dilute  to  500  c.c. 

(2)  Dissolve  178  grams  of  sodium  potassium  tartrate  and  50 
grams  of  sodium  hydroxide  in  water  and  dilute  to  500  c.c. 

Determination.  —  Mix  15  c.c.  of  each  of  the  above  reagents  in  an 
Erlenmeyer  flask  having  a  capacity  of  250  to  300  c.c.,  dilute  with  50 
c.c.  of  freshly  boiled  distilled  water  and  place  the  flask  in  a  boiling 
water-bath  for  five  minutes  ;  then  add  25  c.c.,  accurately  measured 
from  a  burette  or  pipette,  of  a  solution  containing  approximately 
0.5  per  cent,  of  the  sugar  to  be  determined  and  allow  the  mixture 
to  stand  in  the  boiling  water-bath  for  fifteen  minutes.  Remove 
the  flask  from  the  bath  and  filter  as  quickly  as  possible  (using 
moderate  suction)  through  asbestos  prepared  as  described  below  ; 
wash  the  cuprous  oxide  with  boiling  distilled  water  until  the  fil- 
trate is  no  longer  alkaline.  The  cuprous  oxide  can  now  be  (i) 
washed  with  alcohol  and  then  with  ether,  dried  in  a  boiling  water 
oven  for  20  minutes  and  weighed ;  (2)  ignited  and  weighed 
as  cupric  oxide,  as  recommended  by  Defren;  or  (3)  dissolved  in 
nitric  acid  and  the  copper  determined  by  electrolysis  or  by  any 
other  reliable  method  in  which  case  it  will  not  be  necessary  to 
use  asbestos  especially  prepared  by  boiling  with  acid  and  alkali. 
From  the  weight  of  copper  or  cuprous  oxide  determined,  calculate 
the  equivalent  amount  of  cupric  oxide  and  find  the  corresponding 
weight  of  reducing  sugar  from  Defren's  table. 

*Journ.  Amer.  Chem.  Soc.,  1896,  18,  749. 
.  Chem.  Soc.,  1876,  30,  130. 


CARBOHYDRATES—  GENERAL  METHODS.        73 

Precautions.  —  If  the  copper  reduced  is  to  be  weighed  as  cuprous 
or  cupric  oxide  on  the  asbestos  filter,  the  latter  must  be  especially 
prepared  in  order  that  it  shall  lose  no  weight  when  treated  with 
the  hot  alkaline  Fehling  solution.  Asbestos  'of  good  quality  is 
boiled  with  nitric  acid  (1.05  to  i.io  sp.  gr.)  washed  with  water 
then  boiled  with  25  per  cent,  sodium  hydroxide,  washed,  and  the 
treatment  with  acid  and  alkali  repeated.  The  prepared  asbestos  is 
used  to  make  a  tight  felt  about  one  centimeter  thick  in  a  Gooch 
crucible.  When  the  crucible  has  been  prepared  for  use  and 
weighed  it  should  be  tested  by  running  through  it  a  "blank"  of 
hot  alkaline  Fehling  solution  and  washing  with  water  as  in  a  reg- 
ular determination.  The  loss  of  weight  should  not  exceed  one 
half  milligram.  After  each  determination  the  precipitate  is  dis- 
solved in  nitric  acid  and  the  crucible  washed,  ignited  and  re- 
weighed.  If  a  loss  of  over  one  milligram  is  found,  the  determina- 
tion should  be  rejected  and  the  filter  treated  alternately  with  acid 
and  alkali  until  it  ceases  to  lose  in  weight. 

Notes  on  Defreris  Table.  —  Defren  determined  the  amount  of 
copper  reduced  by  fifteen  to  twenty  known  solutions  each  of  dex- 
trose, maltose  and  lactose  with  the  following  results  : 


Dextrose  =  (0.4400  -f  0.000037 
Maltose  =  (0.7215  -f  0.000061 
Lactose  =  (0.6270  +  0.00005  3 


In  which  W  is  the  weight  of  cupric  oxide  obtained,  the  values 
of  ^varying  from  30  to  320  milligrams. 

From  these  formulae  it  is  apparent  that  the  reducing  power  of 
each  of  the  sugars  increases  slightly  with  the  concentration,  as  was 
found  to  be  the  case  in  Soxhlet's  volumetric  method. 

The  table  on  the  following  page  is  based  on  these  equations. 
This  table  is  condensed  from  that  given  in  Defren's  paper  (  /.  c.) 
in  which  the  weights  of  sugars  corresponding  to  each  milligram 
of  cupric  oxide  are  stated.  In  using  either  table  the  weight  of 
copper  or  cupric  oxide  should  be  taken  to  one-tenth  milligram  and 
the  corresponding  weight  of  reducing  sugar  found  by  the  method 
of  proportional  parts. 


74 


ORGANIC   ANALYSIS. 


Defreris  7 able  for  Dextrose,  Maltose  and  Lactose. 


Cupric 
Oxide 
mgms. 

Dextrose 
mgms. 

Maltose 
mgms. 

Lactose 
mgms. 

Cupric 
Oxide 
mgms. 

Dextrose 
mgms 

Maltose 
mgms. 

Lactose 
mgms. 

30 

13.2 

21.7 

18.8 

1  80 

80.4 

I3I.8 

114.6 

40 

I7.6 

29.0 

25.2 

I90 

84.9 

I39-I 

121.  0 

50 

22,1 

36.2 

31-5 

200 

89.5 

146.6 

127.5 

60 

26.5 

43-5 

37-8 

210 

94.0 

I54-I 

I34.I 

70 

3°-9 

50.8 

44.1 

220 

98.6 

161.5 

140.6 

80 

35-4 

58.1 

50.5 

230 

I03.I 

I69.I 

147.0 

90 

39-9 

65-5 

56.8 

240 

107.7 

176.6 

153-5 

100 

44.4 

72.8 

63.2 

250 

II2.3 

184.1 

1  6O.O 

1  10 

48.9 

80.  i 

69-5 

260 

II6.9 

I9I.6 

166.5 

120 

53-3 

87-4 

75-9 

270 

I2I.4 

199.2 

173-0 

130 

57-8 

94.8 

82.4 

280 

I26.I 

206.8 

179.6 

I4O 

62.2 

102.  1 

88.7 

290 

I30-7 

214-3 

186.2 

150 

66.8 

109.5 

95-2 

300 

135-3 

221.9 

192.8 

1  60 

71.3 

116.9 

101.7 

3IO 

139-9 

229.6 

199.3 

170 

75.8 

124.4 

108.2 

320 

144-5 

237-2 

205.9 

KJELDAHL'S  GRAVIMETRIC  METHOD.  * 

As  the  result  of  study  of  many  of  the  conditions  affecting  the 
gravimetric  determination  of  reducing  sugars,  Kjeldahl  recom- 
mended the  following  method : 

Reagents. — (i)  Copper  Solution  :  34.65  grams  of  pure  crystallized 
copper  sulphate  in  500  c.c.  (2)  Alkaline  Solution :  65  grams  of 
sodium  hydroxide,  determined  by  titration,  in  500  c.c.  (3)  Pure 
sodium  potassium  tartrate  pulverized  and  kept  in  the  dry  state. 

Determination.  —  In  an  Erlenmeyer  flask  holding  about  150  c.c., 
mix  5.2  grams  of  sodium  potassium  tartrate  and  15  c.c.  each  of 
the  alkaline  and  copper  solutions,!  add  a  known  volume  of  the 
sugar  solution,  which  must  not  contain  over  0.140  gram  of  dextrose 
or  an  equivalent  amount  of  other  reducing  sugar,  and  fill  with  water 
to  100  c.c.;  close  the  flask  with  a  two-holed  stopper  and  pass  a 
current  of  hydrogen  or  illuminating  gas  until  all  air  is  expelled  from 
the  flask ;  stand  the  flask  in  a  boiling  water  bath  for  exactly  20 
minutes ;  filter,  and  determine  the  reduced  copper  as  described 
under  Defren's  method.  Find  the  corresponding  weight  of  re- 
ducing sugar  from  the  table  on  the  opposite  page. 

* Midelelser  fra  Carlsberg  Lab.,  1895,  5,  I  ;  Abs.  Analyst,  1895,  20,  227  ;  Ztschr. 
anal.  Chem.  1896,  35,  344  ;  Bujard  and  Baier's  Hilfsbuch  fur  Nahrungsmittelchemiker, 
Berlin,  1900. 

finis  gives  practically  30  c.c.  of  the  "mixed  Fehling  reagent"  containing  the 
same  quantity  of  copper  as  is  used  in  Defren's  method.  Kjeldahl' s  method  also  in- 
cludes directions  and  tables  for  the  use  of  15  c.c.  or  50  c.c.  of  the  mixed  copper  reagent 
if  necessary.  The  proportions  here  given  are  adapted  to  such  solutions  as  would  usually 
be  obtained  in  the  estimation  of  reducing  sugars. 


CARBOHYDRATES— GENERAL  METHODS.       75 


KjeldaJiVs  Table  for  Reducing  Sugars. 


Copper 

Dextrose 

Levulose 

Invert-Sugar 

Galactose 

Lactose 

Maltose 

mgms. 

mgms. 

mgms. 

mgms. 

mgms. 

mgms. 

mgms. 

40 

17.8 

19.8 

I9.I 

19.8 

26.7 

30.9 

£ 

22.4 
27.0 

24.9 
3O.I 

23-9 
28.8 

25.0 
3O.I 

33-6 

4O.6 

38.8 
46.8 

E 

3L8 
36.6 

35-2 
40.5 

3.3.8 
38.8 

35-4 
40.7 

47-6 
54-6 

54.8 
63.0 

90 

41.4 

45-9 

43-9 

46.0 

61.8 

71.2 

100 

46.4 

49.1 

51-5 

69.0 

79-4 

no 

51-4 

56^8 

54-4 

57-0 

76.4 

87.7 

120 

56.6 

62.4 

59.8 

62.6 

83.8 

96.1 

130 

61.8 

68.0 

65.2 

68.3 

91.3 

104.7 

140 

67.I 

73-7 

70-7 

74.0 

98.9 

"3-3 

150 

72.5 

79-6 

76.3 

79-9 

106.5 

121.9 

1  60 

78.0 

85-5 

82.0 

85.8 

II4-3 

130.7 

170 

83.7 

9i.5 

87.9 

91.9 

122.2 

139.6 

1  80 

89.4 

97.6 

93-8 

98.0 

130.3 

148.6 

I90 

95-3 

103.9 

99.9 

104.3 

138.4 

157.6 

200 

101.4 

1  10.  2 

106.  i 

1  10.6 

146.7 

166.8 

210 

107-5 

II6.7 

112.4 

117.1 

176.1 

220 

"3-9 

123.2 

II8.8 

123.8 

163.5    . 

185.5 

230 

120.4 

I3O.O 

125-5 

130.5 

172.2 

195-  1 

240 

127.1 

136.8 

132.2 

137.5 

iSl.O 

204.7 

250 

I34-I 

143.8 

139.2 

144-5 

189.9 

214.4 

260 

141.2 

150.9 

146.3 

151.8 

199.0 

224.3 

BARFOED'S  CUPRIC  ACETATE  METHOD.* 

For  the  detection  of  dextrose  Barfoed  used  a  slightly  acid  solu- 
tion of  cupric  acetate  in  place  of  the  Periling  solution.  Lieben  at- 
tempted to  determine  dextrose  quantitatively  in  the  presence  of 
maltose  by  means  of  such  solutions.  This  test,  like  the  formation 
of  the  osazone,  is  not  of  much  quantitative  value  unless  for  com- 
parisons under  identical  conditions,  but  is  often  useful  as  a  quali- 
tative method  for  distinguishing  between  dextrose  (or  other  mono- 
saccharide)  and  those  disaccharides  which  also  reduce  Fehling's 
solution. 

Reagent.  —  Dissolve  45  grams  of  neutral  crystallized  cupric  ace- 
tate in  900  c.c.  of  water,  filter  if  necessary ;  add  1 .2  c.c.  of  50  per 
cent,  acetic  acid  and  dilute  to  a  liter.  A  portion  of  this  reagent 
heated  in  a  water-bath  must  show  no  change. 

Test.  —  To  5  c.c.  of  this  reagent  in  a  test-tube  add  5  c.c.  of  the 
solution  to  be  tested  and  place  in  a  boiling  water-bath  for  3J^  min- 
utes; examine  for  cuprous  oxide,  viewing  the  tube  against  a  black 
background  in  a  good  light.  If  no  evidence  of  reduction  is  found, 

*  Barfoed:  Ztschr.  anal.  CAem.,  1873,  12,  27.  Muller  and  Hagen :  Arch.  ges. 
Physiol.  (Pfluger},  22,  325.  Lieben  :  Ztschr.  Vereins  Rubenzucker- Industrie,  1884, 
34>  857  ;  Wiley,  Agricultural  Analysis,  Vol.  III.,  p.  291. 


76  ORGANIC   ANALYSIS. 

allow  the  tube  to  stand  at  room  temperature  for   10  minutes  and 
examine  again. 

Notes.  —  Under  the  conditions  given  maltose  and  lactose  do  not 
reduce  the  reagent.  Longer  heating  or  the  use  of  a  more  acid 
solution  causes  a  reduction  due  to  the  hydrolysis  of  the  disaccha- 
ride.  In  order  to  prevent  this,  the  solution  may  be  heated  to 
40°  only.  At  this  temperature,  however,  dextrose  reduces  very 
slowly  so  that  the  heating  must  be  continued  for  several  hours. 

Recent  experiments  have  shown  that  the  test  as  described  is 
capable  of  distinguishing  between  dextrose  and  maltose  in  aqueous 
solutions  containing  less  than  0.02  per  cent,  of  the  former  and  at 
least  0.2  per  cent,  of  the  latter,  provided  the  results  are  controlled 
by  check  experiments  made  under  identical  conditions  with  solu- 
tions containing  known  amounts  of  dextrose  and  maltose.  Dextrin 
does  not  interfere.  (Unpublished  results  by  L.  J.  Cohen.) 

If  the  solution  tested  contains  proteids  these  are  precipitated  by 
the  copper,  rendering  the  test  slightly  less  delicate. 

In  applying  the  reaction  to  an  unknown  solution,  comparative 
tests  should  be  made  as  directed  in  the  case  of  the  phenylhydrazine 
test. 

REACTIONS   WITH  ACIDS. 

All  carbohydrates  on  treatment  with  moderately  strong  hydro- 
chloric or  sulphuric  acid  yield  furfurol,  which  is  probably  the  cause 
of  the  color  reaction  with  a-naphthol  described  below.  Hexa- 
carbohydrates  form  only  small  amounts  of  furfurol  and  relatively 
large  amounts  of  levulinic  acid.  Penta-carbohydrates  yield  large 
amounts  of  furfurol  and  no  levulinic  acid.  In  all  cases,  however, 
there  is  more  or  less  formation  of"  humus  "  and  other  by-products. 


MOLISCH'S  a-NAPHTHOL  REACTION. 


Mix  2  to  3  c.c.  of  the  very  dilute  solution  to  be  tested  with  2  or  3 
drops  of  a  15  per  cent,  solution  of  a-naphthal  in  alcohol  or  chloro- 
form, incline  the  tube  and  pour  in  carefully  2  to  3  c.c.  of  pure  con- 
centrated sulphuric  acid.  In  the  presence  of  carbohydrate,  a  violet 
zone  appears  quickly  and  spreads  by  diffusion.  If  the  solution 
tested  contains  more  than  a  few  milligrams  of  carbohydrate,  it 

*Molisch:    Monatsh.  Chem.,  1886,  7,  198. 
Udranszky  :  Ztschr.  physiol.  Chem.,  1888,  12,  355,  377. 
Tollens  :  Handbuch  der  Kohlenhydrate,  II.,  101. 
Mulliken  :  Identification  of  Pure  Organic  Compounds,  I.,  26. 


CARBOHYDRATES— GENERAL  METHODS.        77 

quickly  blackens  and   on   dilution  with  water  gives  a  dull  violet 
precipitate. 

A  similar  test  with  thymol  gives  a  crimson  or  carmine-red  solu- 
tion which  soon  becomes  turbid. 

FURFUROL  TEST  FOR  PENTOSES  AND  PENTOSANS. 

Place  the  substance  to  be  tested  in  an  Erlenmeyer  flask,  add 
hydrochloric  acid  (1.06  sp.  gr.)  and  boil.  Lay  over  the  mouth  of 
the  flask  a  small  filter  paper  moistened,  with  a  solution  of  anilin 
acetate.*  If  the  vapor  escaping  from  the  flask  contains  more  thai* 
traces  of  furfurol  a  bright  red  coloration  appears.  A  test  with 
sucrose  or  pure  starch  will  show  the  amount  of  color  to  be  expected 
from  hexa-carbohydrates. 

If  the  reaction  obtained  from  the  unknown  substance  is  much 
stronger  than  from  sucrose  or  starch,  the  presence  of  pentoses  or 
pentosans  is  indicated.  A  few  other  substances  such  as  glycuronic 
acid  and  oxycellulose  have  been  found  to  yield  considerable 
amounts  of  furfurol,  but  these  rarely  occur  in  sufficient  quantities 
to  require  consideration. 

DETERMINATION  OF  PENTOSES  AND  PENTOSANS. 

Under  carefully  regulated  conditions  the  yield  of  furfurol  from 
either  xylose  or  arabinose,  or  from  the  corresponding  anhydride,  is 
nearly  constant.  If  the  furfurol  is  distilled  and  collected,  its  amount 
can  be  estimated  by  adding  anilin  acetate  and  comparing  the  red 
color  with  that  shown  by  furfurol  solutions  of  known  strength."]"  This 
method  is  delicate,  but  gives  only  approximate  results.  Unless 
the  quantity  is  very  small,  it  should  be  determined  gravimetrically. 

Furfurol  forms  sparingly  soluble  condensation  products  with  a 
number  of  bases  and  phenols.  Phenylhydrazine  and  phloroglucin 
have  been  principally  used  as  precipitants.J  The  Association  of 
Official  Agricultural  Chemists  has  adopted  provisionally  the  phloro- 
glucin method  in  the  following  form  :§ 

Three  grams  of  the  material  are  placed  in  a  flask,  together  with 

*  Prepared  by  mixing  equal  volumes  of  anilin  and  50  per  cent,  acetic  acid. 

f  This  colorimetric  method  is  also  used  for  the  estimation  of  furfurol  in  distilled 
liquors. 

J  Barbituric  acid  has  recently  been  recommended  as  preferable  to  phenylhydrazine 
and  phloroglucin  as  a  precipitant  for  furfurol.  Unger  :  Dissertation,  Munich,  1904. 
Jager  and  Unger  :  Ber.  deut.  chem.  Ges.,  1902,  35,  4440. 

\  Bull.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture.  For  a  review  and  discussion  of 
the  methods  of  determining  the  pentosans  and  the  practical  applications  of  the  results 
see  Tollens,  Krober  and  Rimbach  :  Ztschr.  angew.  Chem.,  1902,  15,  477,  508.  Also 
Fraps  :  Amer.  Chem.  Journ.,  1901,  25,  501. 


78  ORGANIC   ANALYSIS. 

100  c.c.  of  12  per  cent,  hydrochloric  acid  (sp.  gr.  1.06)  and  several 
pieces  of  recently  heated  pumice  stone.  The  flask,  placed  upon 
wire  gauze,  is  connected  with  a  condenser  and  heat  applied,  rather 
gently  at  first,  using  a  gauze  top  to  distribute  the  flame,  which 
is  so  regulated  as  to  distill  30  c.c.  in  about  ten  minutes.  The  30 
c.c.  driven  over  are  replaced  by  a  like  quantity  of  the  dilute  acid, 
.and  the  process  continued  so  long  as  the  distillate  gives  a  pro- 
mounced  reaction  with  anilin  acetate  on  filter  paper.  To  the  com- 
pleted distillate  is  gradually  added  a  quantity  of  phloroglucin  (free 
from  diresorcin)  dissolved  in  12  per  cent,  hydrochloric  acid,  and  the 
resulting  mixture  thoroughly  stirred.  The  amount  of  phloroglucin 
used  should  be  about  double  that  of  the  furfurol  expected.  The  solu- 
tion turns  first  yellow,  then  green  and  very  soon  an  amorphous 
greenish  precipitate  appears,  which  grows  rapidly  darker,  till  it 
finally  becomes  almost  black.  The  solution  is  made  up  to  500  c.c. 
with  12  percent,  hydrochloric  acid,  and  allowed  to  stand  over  night. 
The  amorphous  black  precipitate  is  filtered  into  a  tared  gooch 
through  an  asbestos  felt,  washed  with  100  c.c.  of  water,  dried  to 
constant  weight  by  heating  from  three  to  four  hours  at  100°, 
cooled  and  weighed,  the  increase  in  weight  being  reckoned  as 
phloroglucid.  To  calculate  the  furfurol  from  the  phloroglucid,  the 
following  formulae  are  used  : 

Phloroglucid  (less  than  0.2  gram)  -=-  1.82  =  furfurol. 
Phloroglucid  (from  0.2  to  0.3  gram)  -=-  1.895  =  furfurol. 
Phloroglucid  (from  0.3  to  0.4  gram)  -=-  1.92  =  furfurol. 
Phloroglucid  (above  0.4  gram)  -r-  1.93  =  furfurol. 

To  the  weight  of  furfural  thus  found,  0.0104  gram  is  added  as  a 
correction  for  solubility  and  the  corrected  weight  is  multiplied  by 
1.91  for  xylose,  2.35  for  arabinose  or  2.13  for  "  pentose." 

Qualitative  test  of  the  purity  of  the  phloroglucin. —  Dissolve  a  small 
quantity  of  the  phloroglucin  in  a  few  drops  of  acetic  anhydrid,  heat 
almost  to  boiling  and  add  a  few  drops  of  concentrated  sulphuric 
acid.  A  violet  color  indicates  the  presence  of  diresorcin.  A  phloro- 
glucin which  gives  more  than  a  faint  coloration  must  be  rejected. 

LEVULINIC  ACID  REACTION  OF  HEXA-CARBOHYDRATES. 

According  to  Wehmer  and  Tollens,*  all  hexa-carbohydrates 
yield  levulinic  acid  in  sufficient  quantity  for  identification  when 
treated  as  follows : 

*  Ann.  Chcm.,  1887,  243,  314. 


CARBOHYDRATES— GENERAL  METHODS.        79 

Heat  5  to  20  grams  of  the  substance  with  IOO  c.c.  of  hydro- 
chloric acid  (i.io  sp.  gr.)  for  18  hours  on  a  boiling  water-bath; 
filter  the  solution,  shake  with  ether  to  extract  the  levulinic  acid, 
and  convert  the  latter  into  the  zinc  or  silver  salt  for,  identification. 
As  this  method  is  purely  qualitative  and  is  not  often  used  in  ordi- 
nary analytical  work,  reference  must  be  made  to  the  original  paper 
for  details  of  manipulation. 

OXIDATION  BY  NITRIC  ACID. 

By  heating  with  moderately  strong  nitric  acid,  xylose  and  ara- 
binose  are  oxidized,  each  yielding  a  trioxyglutaric  acid,  COOH- 
(CHOH)3COOH,  while  aldoses  of  the  hexose  group  yield  the 
corresponding  acids,  COOH(CHOH)4COOH,  saccharic,  man- 
nosaccharic  and  mucic  acids  being  obtained  respectively  from 
dextrose,  mannose  and  galactose. 

All  of  these  oxidation  products  are  freely  soluble  except  mucic 
acid,  which  is  practically  insoluble  in  water  or  dilute  nitric  acid. 
Since  mucic  acid  is  produced  in  fairly  constant  proportion,  its  in- 
solubility affords  a  means  for  the  approximate  determination  of 
galactose  or  any  substance  which  yields  galactose  on  hydrolysis. 

Mucic  Acid  Method  for  Galactose,  Lactose,  Raffinose  and  G aloe  tans.* 

Weigh  i  to  3  grams  of  substance  according  to  the  amount  of 
mucic  acid  expected ;  remove  fat  if  necessary  by  washing  with 
ether;  transfer  to  a  beaker  about  5.5  cm.  in  diameter  and  7  cm. 
deep;  add  60  c.c.  of  nitric  acid  of  1.15  sp.  gr.  and  evaporate  the 
solution  to  exactly  one  third  its  volume  on  a  water-bath  at  a  tem- 
perature of  94°  to  96°.  After  standing  24  hours,  add  10  c.c.  of 
water  to  the  precipitate  and  allow  it  to  stand  another  24  hours. 
The  mucic  acid  has  now  crystallized,  and,  unless  contaminated 
with  insoluble  residue  from  the  sample,  it  can  be  transferred  to  a 
weighed  filter,  washed  with  30  c.c.  of  water,  then  with  alcohol  and 
ether,  dried  at  100°  and  weighed. 

In  case  other  insoluble  substances  are  present,  return  the  filter 
and  mucic  acid,  after  washing  with  water,  to  the  beaker;  warm  15 
minutes  with  a  mixture  of  I  part  strong  ammonia,  I  part  ammo- 
nium carbonate  and  19  parts  of  water  ;  filter  and  wash;  evaporate 
filtrate  to  dry  ness  over  a  water-bath  ;  add  5  c.c.  nitric  acid  of  1.15  sp. 

*Tollens  and  Rischbiet :  Bet.  deut.  chem.  Ges.,  1885,  18,  2616.  Creydt :  Ibid., 
1886,  19,  3115.  Bull.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


8o  ORGANIC   ANALYSIS. 

gr. ;  stir  thoroughly  and  allow  to  stand  for  30  minutes.  Collect 
the  mucic  acid  on  a  weighed  filter,  wash  with  10  to  15  c.c.  of  water, 
then  with  60  c.c.  of  alcohol  and  a  number  of  times  with  ether ;  dry 
at  1 00°  and  weigh. 

When  these  directions  are  strictly  followed  galactose  yields  about 
three  fourths  its  weight  of  mucic  acid.  The  weight  of  mucic  acid 
obtained  from  lactose,  raffinose  or  galactan  is  calculated  as  three 
fourths  the  weight  of  galactose  which  could  be  obtained  by  hydroly- 
sis. The  yield  of  mucic  acid  is,  however,  considerably  influenced 
by  the  details  of  manipulation.  .In  carrying  out  the  method  a 
comparative  determination  with  pure  milk  sugar  should  always  be 
made,  as  substances  may  be  present  which  prevent  the  crystalliza- 
tion of  the  mucic  acid.* 

HYDROLYSIS  BY  DILUTE  ACIDS. 

Monosaccharides,  as  the  name  implies,  cannot  be  hydrolyzed  to 
simpler  sugars.  As  a  rule,  they  are  unaffected  by  dilute  acids  ex- 
cept on  prolonged  heating,  when  they  are  gradually  attacked, 
yielding  in  part  decomposition  products  like  those  produced  by 
stronger  acids,  and  undergoing  a  partial  "  reversion  "  with  the  for- 
mation of  di-  or  polysaccharides.  Dextrose,  for  example,  when 
heated  too  long  with  dilute  hydrochloric  or  sulphuric  acid,  is  changed 
partially  to  "  isomaltose  "  and  to  dextrin-like  anhydrides,  such  as 
"gallisin,"  the  unfermentable  constituent  of  crude  commercial  glu- 
cose. Levulose  decomposes  much  more  readily  than  dextrose  on 
heating  with  dilute  acids. 

Disaccharides  differ  considerably  in  the  readiness  with  which  they 
are  hydrolyzed  by  acids.  Sucrose  is  very  easily  hydrolyzed,  a  20 
per  cent,  solution  being  completely  changed  to  invert-sugar  by 
mixing  with  one-tenth  its  volume  of  concentrated  hydrochloric  acid 
(making  about  3  per  cent,  of  actual  acid  in  the  mixture)  and  warm- 
ing to  68°  at  such  a  rate  as  to  require  15  minutes  heating.f 

Maltose  is  less  easily  hydrolized  than  sucrose,  a  2  per  cent, 
solution  in  2  to  3  per  cent,  hydrochloric  acid  requiring  30  to  40 
minutes'  boiling,  or  2  to  3  hours'  heating  on  a  water  bath,  for  com- 

*Herzfeld:  Ztschr.  Vereins  Robenzucker  Ind.,  1890,  40,  265;  Abs.  Ghent.  Ztg.> 
1890,  14  Rep.)  108.  Stone  and  Baird  \Jour.  Amer.  Chem.  Soc.,  1897,  19,  119. 

f  Clerget's  method,  described  by  Wiley:  Agricultural  Analysis,  Vol.  III.,  pp. 
105—107.  According  to  Borntrager  and  to  Samelson  {Ztschr.  angeiu.  Chem'.,  1892, 
334  ;  1893,  690  ;  1894,  267,  351),  the  sucrose  in  such  a  mixture  is  completely  hydro- 
lyzed by  standing  over  night  at  room  temperature. 


CARBOHYDRATES—  GENERAL  METHODS.        81 

plete  hydrolysis  to  dextose.*  Lactose  is  also  less  readily  hydro- 
lyzed  than  sucrose,  the  difference  being  especially  marked  in  the 
case  of  weak  acids.  By  boiling  with  citric  acid,  sucrose  can  be 
completely  converted  to  invert-sugar,  while  lactose  remains  un- 
changed. (See  methods  for  sucrose  and  lactose  given  later.) 

Raffinose  is  hydrolyzed  by  dilute  mineral  acids,  slowly  in  the 
cold,  much  more  rapidly  on  boiling,  requiring  in  either  case  more 
vigorous  treatment  than  does  sucrose. 

Dextrin,  glycogen  and  starch  are  hydrolyzed  to  dextrose  under 
the  same  conditions  as  maltose,  but  require  somewhat  longer  heat- 
ing. Pentosans  and  galactans  (at  least  the  more  common  forms) 
are  hydrolyzed  by  acids  almost  as  readily  as  starch.  Normal  cel- 
lulose is  not  hydrolyzed  by  boiling  dilute  acids.  Hemicellulose  is 
a  term  commonly  applied  to  the  carbohydrate  matter  in  the  cell 
walls  of  plants,  more  resistant  to  enzymes  than  starch,  but  dis- 
solved and  hydrolyzed  on  boiling  with  dilute  mineral  acids.  The 
hemicellulose  of  the  common  food  plants  consists  largely  of 
pentosans. 

ROTATION   OF   POLARIZED    LIGHT. 

Solutions  of  the  natural  carbohydrates  have  the  property  of  rota- 
ting the  plane  of  polarized  light.  This  rotating  power  is  also 
shown  by  most  other  natural  substances  containing  one  or  more 
asymmetric  carbon  atoms  in  the  molecule. 

MEASURE  OF  ROTATING  POWER  —  SPECIFIC  ROTATION. 

The  first  organic  substances  to  be  studied  by  means  of  polarized 
light  were  liquids,  or  solutions  of  known  density.  In  order  to  re- 
duce the  observations  to  a  common  basis  for  comparison  Biot  intro- 
duced the  following  formula  : 


in  which  [«]  =the  "specific  rotating  power  " 

a  =  the  observed  angular  degrees  of  rotation 

/=the  length  in  decimeters  of  the  column  of  liquid 

traversed  by  the  polarized  light 
df=the  density  of  the  liquid. 

*  It  is  sometimes  stated  that  maltose  cannot  be  made  to  yield  more  than  about  98 
per  cent,  of  the  theoretical  amount  of  dextrose,  the  acid  always  beginning  to  attack  the 
latter  before  the  hydrolysis  of  the  former  is  quite  complete.  See  notes  on  the  deter- 
mination of  starch. 


82  ORGANIC   ANALYSIS. 

On  extending  the  calculation  to  solids    in  solution  the  above 
formula  becomes 

100  a 


in  which  /  =  the  percentage  of  solid  in  the  solution. 

But  since  d  x  /  equals  the  number  of  grams  of  solid  in  100  c.c. 
of  solution  it  is  simpler  to  write  the  formula 

100  a 

[«]  =  -j— 
I  x  c 

in  which  c=  concentration,  t.  e.,  the  number  of  grams  of  dissolved 
solid  in  100  c.c.  of  the  solution. 

The  value  of  the  specific  rotating  power  [a]  is,  therefore,  the 
number  of  angular  degrees  through  which  a  ray  of  polarized  light 
would  be  rotated  in  traversing  one  decimeter  of  a  solution  of  which 
each  cubic  centimeter  contained  one  gram  of  the  active  substance. 

The  angular  rotation  is  always  directly  proportional  to  the  length 
of  the  column  of  liquid  through  which  the  light  passes.  It  always 
depends  upon  the  wave  length  of  the  light  ray  employed.  By 
measuring  the  rotation  for  different  rays  the  rotation-dispersion  of 
the  substance  is  determined.  Sodium  light  corresponding  to  the 
Fraunhofer  line  D  is  commonly  used  in  determining  the  rotating 
power.  In  many  cases  the  rotating  power  is  appreciably  influenced 
by  temperature.  The  latter  should  therefore  be  stated  in  giving 
the  value  of  [a].  Thus  [a]/0  indicates  the  rotating  power  as  meas- 
ured with  D  light  at  20°.  The  concentration  of  the  solution  has 
usually  an  appreciable  influence  upon  the  value  of  [a]  as  is  shown 
by  the  following  statements  of  rotating  power  of  the  more  import- 
ant carbohydrates: 

Dextrose  (Tollens)* 

[«]1>17=t  52.50  -f  o.o  1  8796^  +  0.0005  168/2. 
Levulose  (Jungfleish  and  Grimbert)  \ 

\oi\jf  =  —  [101.38  —  0.56^+  o.  io8(V  —  10)]. 
Galactose  (Meissl)§ 

\_a~\j*  =  83.883  -f  0.0785^  —  0.209*. 

*  Ber.  deut.  chem.  Ges.,  1876,  g,  487,  1531  ;  1884,  17,  2234. 

f  Unless  otherwise  stated  the  rotation  is  to  the  right  (-f);  Isevorotation  is  indciated 

by  (-)• 

J   Compt.  rend.,  1888,  107,  390. 

§  Journ.  prakt.  Chem.,  1880,  [2],  22,  97. 


CARBOHYDRATES— GEXERAL  METHODS.        83 

Sucrose  (Tollens)  * 

[«]/)20  =  66.386  4-  0.015035;)  —  o.ooo3986/>2. 
Lactose  (Schmoger)f 

[a]Z)20=  52.53  (constant  for  c  =  2.4  to  40). 
Maltose  (Meissl)J 

[«]^=  140.375  -o.oi837/-  0.095^. 

At  ordinary  concentrations  and  laboratory  temperatures,  the 
values  for  \_a]D  are  approximately  : 

Dextrose  53  Sucrose  66.5 

Levulose          —[102  —  .56/1  Lactose  52.5 

Invert-sugar — [27-9  —  -320  Maltose  138. 

Galactose  80.5  Raffinose  104.5 

^Ian"ose  '3  ftarch       >  ,90  to  210 

Arabmose  104  Glycogen  ^ 

Xylose  19  Dextrins  160  to  210 

PREPARATION  OF  SOLUTIONS  FOR  POLARIZATION. 

Reducing  sugars  when  first  dissolved  often  show  rotating  powers 
varying  greatly  from  those  given.  This  property,  known  as  mul- 
tirotation,  or  birotation,  occasionally  causes  errors  in  analytical 
work.  The  normal  rotation  is  established  by  allowing  the  solution 
to  stand  for  some  hours,  by  boiling,  or  by  the  addition  of  about 
O.I  per  cent,  of  ammonia.  Solutions  containing  ammonia  darken 
on  standing  and  should  therefore  be  polarized  as  soon  as  prepared. 

Sucrose  does  not  show  multirotation,  and  is  not  appreciably 
affected  by  ordinary  changes  of  temperature,  while  the  changes  of 
rotating  power  due  to  variations  in  concentration  are  small  and 
have  been  accurately  determined. 

Solutions  for  polarization  must  be  clear  and  free  from  all  impuri- 
ties having  an  influence  upon  polarized  light.  Among  the  optically 
active  substances  most  likely  to  be  met  in  natural  products  are  or- 
ganic acids  (especially  tartaric),  pectin  bodies,  gums,  resins,  color- 
ing matters,  glucosides,  alkaloids  and  proteids.  In  most  cases  the 
interfering  substances  can  be  precipitated  and  the  solution  clarified 
by  adding,  first,  basic  lead  acetate,  and  then  a  cream  of  aluminium 
hydroxide.  It  is  sometimes  advantageous  to  add  a  small  amount 
of  tannin  before  clarifying.  Tannin  combines  with  many  of 
the  substances  mentioned,  either  precipitating  them  or  forming 

*  Ber.  deut.  Chem.  Geo.,  1877,  10,  1403. 

f  Ibid.t  1880,  13,  1922. 

J  Journ.  prakt.  Chem.,  1882,  [2],  25,  114. 


84  ORGANIC   ANALYSIS. 

compounds  more  easily  precipitated  by  the  basic  acetate.  In  case 
the  solution,  after  being  treated  with  clarifying  agents,  made  up  to 
definite  volume  and  filtered,  is  too  highly  colored  for  examination 
in  the  polariscope,  it  may  be  poured  slowly  through  a  small  amount 
of  pure  dry  animal  charcoal  on  a  paper  filter.  Since  the  charcoal 
absorbs  a  small  amount  of  the  sugar,  the  first  portions  of  the  filtrate 
must  be  rejected. 

DETERMINATION  OF  ANGULAR  ROTATION. 

For  the  direct  determination  of  \_u-~\D,  sodium  light  must  be  used. 
The  instrument  most  commonly  employed  is  the  Laurent  polari- 
scope. This  has  a  circular  scale  graduated  in  angular  degrees. 
The  scale  is  divided  into  half-degrees  and  the  vernier  is  graduated 
to  read  minutes  of  angular  rotation.  With  this  polariscope  the 
rotation  produced  by  the  active  substance  is  found  by  turning  the 
analyzing  prism,  which  is  fixed  in  position  with  regard  to  the  scale 
but  rotates  on  its  axis.  The  zero  point  is  found  with  the  polari- 
scope empty  and  is  the  point  at  which  the  crystal  axis  of  the 
analyzing  prism  is  at  right  angles  to  the  plane  of  polarization.  If 
now  an  active  substance  is  placed  between  the  polarizing  and 
analyzing  prisms,  the  latter  must  be  rotated  until  it  stands  at  right 
angles  to  the  new  plane,  when  the  appearance  of  the  field  will  be 
the  same  as  that  previously  observed  at  the  zero  point.  The 
position  of  the  scale  now  shows  directly  the  number  of  angular 
degrees  through  which  the  plane  of  polarization  has  been  rotated 
by  the  active  substance. 

For  the  examination  of  sugar  solutions,  especially  in  technical 
analysis,  the  Schmidt  and  Haensch  saccharimeter  with  Ventzke 
scale  is  commonly  used.  This  instrument,  which  is  made 
in  several  forms,  is  different  in  principle  from  the  Laurent 
polariscope.  White  light  is  used  and  the  rotation,  instead  of  being 
measured  directly,  is  "  compensated"  by  means  of  quartz  wedges, 
the  thickness  of  quartz  required  to  neutralize  the  effect  of  the  active 
substance  giving  a  measure  of  the  rotation  produced  by  the  latter. 
With  the  trough  of  the  saccharimeter  empty,  the  scale  should  read 
zero  when  the  quartz  wedges  are  so  placed  that  the  light  passes 
through  equal  thicknesses  of  positive  (dextrorotatory)  and  nega- 
tive (laevorotatory)  quartz.  If  now  a  tube  containing  a  solution  of 
sucrose  is  introduced,  the  thickness  of  negative  quartz  is  increased, 
or  that  of  positive  quartz  diminished,  until  the  rotation  produced 


CARBOHYDRATES— GENERAL  METHODS.        85 

by  the  sugar  solution  is  compensated  by  the  opposite  rotation  of 
the  quartz.  This  method  of  measuring  the  rotation  by  means  of 
quartz  with  white  light  illumination  is  applicable  only  to  such 
active  bodies  as  have  practically  the  same  rotation  dispersion  as 
quartz.  This  is  true  of  aqueous  sugar  solutions,  which  can  there- 
fore be  examined  by  either  of  the  instruments  mentioned.  The 
readings  are  compared  by  assuming  that  each  degree  of  the 
Ventzke  sugar  scale  is  equivalent  to  0.3468  degrees  of  angular 
rotation. 

For  descriptions  of  apparatus  and  details  of  testing  and  manip- 
ulation, the  standard  works  on  sugar  analysis  should  be  consulted. 
A  number  of  the  more  important  of  these  are  among  the  refer- 
ence books  listed  below,  but  attention  may  be  especially  directed 
to  the  work  of  Landolt  as  containing  the  most  exhaustive  and 
authoritative  discussion  of  polarized  light  and  its  analytical 
applications. 

REFERENCES. 

Allen :  Commercial  Organic  Analysis,  Volume  I.,  Philadelphia, 
1898. 

Cross  and  Bevan :  Cellulose,  an  Outline  of  the  Chemistry  of  the 
Structural  Elements  of  Plants,  London,  1898.  Researches  on 
Cellulose,  1895-1900,  London,  1901. 

Effront :  Enzymes  and  Their  Applications,  Volume  I.,  The 
Enzymes  of  the  Carbohydrates  (Translated  by  Prescott),  New 
York,  1902. 

Hoppe-Seyler :  Physiologisch-  und  Pathologisch-Chemischen 
Analyse  (Revised  by  Theirfelder),  Berlin,  1903. 

Landolt:  Optical  Rotation  of  Organic  Substances  (English  edi- 
tion by  Long),  Easton,  Pa.,  1902. 

Lippmann:  Chemie  der  Zuckerarten,  Braunschweig,  1904. 

Maquenne :  Les  Sucres,  Paris,  1900. 

Meyer  and  Jacobson :  Organische  Chemie,  Erster  Band,  1893. 

Milliken:  Identification  of  Pure  Organic  Compounds,  Volume 
I.,  New  York,  1904. 

Sidersky:  Traite  d' Analyze  des  Matieres  Sucrees,  Paris,  1890. 

Tollens:  Chemie  der  Kohlenhydrate,  Band  I.,  1888;  Band  II., 
1895,  Breslau. 

Vaubel :  Bestimmung  Organischer  Verbindungen,  Berlin,  1902. 

Wiley:  Agricultural  Analysis,  Vol.  III.,  Easton,  Pa.  1897. 


CHAPTER  VI. 
Carbohydrates — Special  Methods. 

ANALYSIS    OF    RAW    SUGAR. 
POLARISCOPIC   EXAMINATION. 

A  special  room  of  even  temperature  which  can  be  darkened 
when  desired  should  be  provided  for  the  polariscopic  examina- 
tion. For  illumination  of  the  white-light- saccharimeter,  a  triple 
flame  or  an  Argand  burner  with  incandescent  mantle  is  used.  In 
taking  readings  the  polariscope  must  be  brought  only  near  enough 
to  the  burner  to  secure  a  good  illumination  of  the  field,  never 
within  less  than  eight  inches.  As  soon  as  the  reading  is  taken 
the  polariscope  should  be  turned  away  or  the  flame  lowered,  in 
order  to  avoid  any  possible  warming  of  the  instrument. 

Determination  of  the  Zero  Point. — The  trough  of  the  saccharim- 
eter being  empty,  set  the  scale  within  a  few  degrees  of  zero  and 
focus  the  eyepiece  so  that  the  field  of  vision  is  clear  and  the  per- 
pendicular line  or  band  dividing  it  is  perfectly  distinct.  Now  ro- 
tate the  milled  head  so  as  to  move  the  zero  point  of  the  scale 
toward  that  of  the  vernier.  When  the  neutral  point  (the  true  zero 
point  for  the  instrument  as  it  stands)  is  reached  the  appearance  of 
the  entire  field  is  uniform.  Approach  the  zero  point  first  from  one 
side  then  from  the  other,  taking  the  reading  each  time  as  soon  as 
the  field  appears  uniform,  until  successive  readings  do  not  differ  by 
more  than  0.2°  on  the  Ventzke  sugar  scale.  The  average  of  ten 
such  readings  is  taken  as  the  zero  point  in  all  subsequent  work 
with  the  instrument  unless  it  should  be  jarred  or  moved,  in  which 
case  the  zero  point  should  be  redetermined. 

lest  with  Pure  Sugar. — Weigh  26*  grams  of  pure  sucrose,  dis- 

*  The  value  of  the  Ventzke  scale  was  originally  fixed  by  means  of  pure  sucrose 
solutions  of  1. 100  sp.  gr.  at  17.5°  and  it  was  found  that  loo  c.c.  of  such  a  solution 
contain  26.048  grams  of  sucrose  (weighed  in  air  with  brass  weights) .  For  many  years, 
however,  it  has  been  the  custom  of  instrument  makers  to  calibrate  the  Ventzke  scale 
by  means  of  solutions  containing  26.048  grams  of  sucrose  in  loo  Mohr  cubic  centi- 
meters. A  solution  of  very  nearly  the  same  strength  is  obtained  by  dissolving  26 
grams  and  completing  the  volume  at  20°  to  too  metric  cubic  centimeters  (the  volume 
occupied  by  100  grams  of  water  at  4°  weighed  in  vacuo).  The  latter  proportions  have 
recently  been  adopted  by  the  International  Commission  for  Unifying  Methods  of  Sugar 
Analysis. 

86 


CARBOHYDRATES— SPECIAL  METHODS.         87 

solve  in  water  in  an  accurately  calibrated  100  c.c.  flask,  fill  to  the 
mark  at  20°  to  22°  and  mix  throughly  by  shaking,  holding  the 
flask  in  such  a  way  as  not  to  warm  the  solution.  The  latter  should 
have  a  density  very  nearly  i.io  and  should  rotate  the  plane  of 
polarized  light  34.68°  to  the  right,  giving  a  reading  of  100.0° 
on  the  Ventzke  scale. 

Rinse  the  200  mm.  tube  several  times  with  the  solution,  then  fill 
until  the  curved  surface  of  the  liquid  projects  above  the  open  end 
of  the  tube  ;  see  that  all  air-bubbles  have  risen  to  the  surface  and 
then  slide  on  the  cover-glass  horizontally  in  such  a  manner  that 
the  excess  of  liquid  is  carried  over  the  side,  leaving  the  cover-glass 
exactly  closing  the  tube,  with  no  air-bubbles  beneath  it  and  none 
of  the  liquid  upon  its  upper  surface.  The  cover-glass  being  in  po- 
sition, the  tube  is  closed  by  screwing  on  the  cap.  The  latter 
should  be  only  tight  enough  to  prevent  leakage,  as  any  consider- 
able pressure  on  the  glass  plate  may  cause  it  to  become  optically 
active. 

Place  the  tube  in  the  trough  of  the  saccharimeter  or  polariscope 
and  take  ten  readings  as  in  setting  the  zero  point.  The  average 
reading  (corrected  for  zero  point)  should  not  differ  from  100.0° 
by  more  than  0.2°  on  the  Ventzke  scale,  nor  from  34°4i'  on  the 
Laurent  polariscope  by  more  than  5'. 

Polarization  of  the  Razv  Sugar.  —  Mix  the  sample,  weigh  out  26 
grams  and  dissolve  in  60  to  80  c.c.  of  water  in  a  100  c.c.  flask. 
When  the  sugar  is  entirely  dissolved,  add  from  I  to  5  c.c.  (accord- 
ing to  the  nature  of  the  sample ;  only  very  dark  sugars  should  re- 
quire more  than  2  c.c.)  of  a  solution  of  basic  lead  acetate  of  about 
1.25  sp.  gr.*  A  decided  excess  of  basic  acetate  should  never  be 
used.  After  the  lead  acetate  has  been  added  and  mixed,  add  twice 
its  volume  of  "  alumina  cream."  f  This  assists  in  the  clarification, 
precipitates  the  excess  of  lead,  and  facilitates  filtration.  A  moder- 
ate excess  of  alumina  cream  does  no  harm.  With  high  grade 

*This  may  be  prepared  by  dissolving  the  solid  basic  salt  or  by  boiling  an  excess 
of  litharge  with  a  strong  solution  of  neutral  lead  acetate. 

f  Prepared  as  follows  :  Shake  powdered  commercial  alum  with  water  at  ordinary 
temperature  until  a  saturated  solution  is  obtained.  Set  aside  a  little  of  the  solution, 
and  to  the  residue  add  ammonia,  little  by  little,  stirring  between  additions,  until  the 
mixture  is  alkaline  to  litmus  paper.  Then  drop  in  additions  of  the  portion  left  aside, 
until  the  mixture  is  just  acid  to  litmus  paper.  By  this  procedure  a  cream  of  aluminium 
hydroxide  is  obtained  suspended  in  a  solution  of  ammonium  sulphate.  This  sul- 
phate is  advantageous  when  added  after  the  basic  acetate  since  it  precipitates  what- 
ever excess  of  lead  may  be  present. 


88  ORGANIC   ANALYSIS. 

sugars  the  use  of  alumina  cream  alone  may  be  sufficient  for  clarifi- 
cation. Make  up  to  volume  with  distilled  water,*  shake  well  and 
then  pour  the  whole  solution,  or  as  much  as  practicable,  on  a  dry 
filter.  Reject  the  first  20  or  30  c.c.  of  filtrate  and  then  polarize 
the  remainder  as  already  described  for  the  pure  sugar  solution 
The  average  reading  of  the  Ventzke  scale,  corrected  for  the  devi- 
ation of  the  zero-point,  is  reported  as  "  polarization." 

Notes  and  Precautions.  —  Care  must  be  taken  to  avoid  the  follow- 
ing errors  due  to  manipulation  ;  (i)  change  in  moisture  content  of 
sample  during  weighing,  (2)  change  in  volume  of  solution  due  to 
fluctuations  of  temperature,  (3)  imperfect  mixing  of  solution  after 
diluting  to  volume  in  the  graduated  flask,  (4)  evaporation  during 
filtration,  (5)  too  great  compression  of  cover  glasses  in  closing  the 
polariscope  tube. 

Clarification  is  very  important.  All  proteids  are  laevoratory  and 
must  be  entirely  removed.  The  solution  must  be  free  from  tur- 
bidity, but  not  necessarily  free  from  color.  Only  so  much  of  the 
clarifying  agents  should  be  used  as  is  necessary  to  free  the  solu- 
tion from  optically  active  impurities  and  from  turbidity.  Any  ex- 
cess increases  the  error  due  to  the  presence  of  precipitate  when  the 
solution  is  diluted  to  volume.  The  volume  occupied  by  the  pre- 
cipitate varies  between  0.05  and  i.o  c.c.  for  ordinary  raw  sugars. 
It  can  be  determined  by  Scheiber's  method  of  double  dilution  t 
in  which  a  duplicate  determination  is  made  in  a  flask  of  twice 
the  volume,  or  by  determining  the  weight  and  density  of  the  pre- 
cipitate as  recommended  by  Sachs,  f  The  latter  method  is  pre- 
ferred by  Wiechmann  §  and  Home  ||  each  of  whom  determined  the 
volume  of  the  precipitate  for  a  number  of  faw  sugars  from  different 
localities.  Home  has  found  (loc.  cit.}  that  the  error  can  be  almost 
entirely  avoided  by  clarifying  with  anhydrous  basic  acetate 
after  the  sugar  solution  has  been  diluted  to  volume. 

Regulations  of  the  International  Commission. 

The  following  resolutions  \  adopted  by  the  International  Com- 
mission for  the  Unification  of  Sugar  Analysis  in  1900,  have  since 
been  generally  accepted. 

*  If  frothing  interferes  at  this  point,  add  two  or  three  drops  of  ether. 
•\Ztschr.   Vereins.  Rubenzucker-Industrie,  1875,  25,  1054. 
\  Ibid.,  1880,  50,  229.     See  also  the  paper  by  Home. 
§  Ibid.,  1903,  [n.  f.]40,  498  ;  Abs.  Journ.  Chem.  Soc.,  1903,  84,  ii,  699. 
\Journ.  Amer.  Chem.  Soc.,  1904,  26,  186. 

^f  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bull.  73,  p.  58.  Journ.  Amer.  Chem.  Soc., 
I9OI>  23,  59. 


CARBOHYDRATES— SPECIAL  METHODS.         89 

1.  In  genera],  all  sugar  tests  shall  be  made  at  20°  C. 

2.  The  graduation  of  the  saccharimeter  shall  be  made  at  20°  C. 
Twenty-six  grams  of  pure  sugar,  dissolved  in  water,  and  the  vol- 
ume made  up  to  100  metric  cubic  centimeters,  or  during  the  period 
of  transition  26.048  grams  of  pure  sugar  in  100  Mohr  cubic  centi- 
meters, all  weighings  to  be  made  in  air  with  brass  weights,  the 
completion  of  the  volume  and  the  polarizations  to  be  made  at  20° 
on  an  instrument  graduated  at  20°,  should  give  an  indication  of 
100  on  the  scale  of  the  saccharimeter.     For  countries  where  tem- 
peratures are  usually  higher  than  20°,  it  is  permissible  that  sacchar- 
imeters  be  graduated  at   30°,  or  any  other  suitable  temperature, 
under  the  conditions  specified  above,  providing  that  the  analysis 
of  the  sugar  be  made  at  the  same  temperature  —  that  is,  that  the 
volume  be  completed  and  the  polarization  made  at  the  temper- 
ature specified. 

3.  Preparation  of  pure  sugar:  Purest  commercial  sugar  is  to  be 
further  purified  in  the  following  manner :  A  hot  saturated  aqueous 
solution  is  prepared  and  the  sugar  precipitated  with  absolute  ethyl 
alcohol ;  the  sugar  is  carefully  spun  in  a  small  centrifugal  machine 
and  washed  in  the  latter  with  absolute  alcohol.     The  sugar  thus 
obtained  is  re-dissolved  in  water,  the  saturated  solution  again  pre- 
cipitated with  alcohol  and  washed  as  above.     The  product  of  the 
second  crop  of  crystals  is  dried  between  blotting  paper  and  pre- 
served in  glass  vessels  for  use.     The  moisture  still  contained  in  the 
sugar  is  determined  and  taken  into  account  when  weighing  the 
sugar  which  is  to  be  used. 

The  committee  further  decided  that  central  stations  shall  be  des- 
ignated in  each  country  which  are  to  be  charged  with  the  prepa- 
ration and  distribution  of  chemically  pure  sugar.  Wherever  this 
arrangement  is  not  feasible,  quartz  plates,  the  values  of  which  have 
been  determined  by  means  of  chemically  pure  sugar,  shall  serve 
for  the  control  of  the  saccharimeters. 

The  committee  further  decided  that  the  above  control  of  quartz 
plates  by  means  of  chemically  pure  sugar  should,  as  a  rule,  apply 
only  to  the  central  stations  which  are  to  test  the  correctness  of 
saccharimeters  ;  for  those  who  execute  commercial  analyses,  the 
repeated  control  of  the  instruments  is  to  be  accomplished,  now  as 
before,  by  quartz  plates. 

4.  In  effecting  the  polarization  of  substances  containing  sugar, 
half-shade  instruments,  or  triple  field,  only  are  to  be  employed. 


90  ORGANIC   ANALYSIS. 

5.  During  the  observation  the  apparatus  must  be  in  a  fixed  posi- 
tion and  so  far  removed  from  the  source  of  light  that  the  polarizing 
nicol  is  not  warmed. 

6.  Sources    of  light    may  be   gas,  triple   burner  with  metallic 
cylinder,  lens    and   reflector;    gas   lamp   with    Auer  (Welsbach) 
burner ;    electric   lamp ;    petroleum    duplex   lamp ;    sodium    light. 
Several  readings  are  to  be  made  and  the  mean  thereof  taken,  but 
any  one  reading  must  not  be  neglected. 

7.  In  making  a  polarization  the  whole  normal  weight  for   100 
cubic  centimeters  is  to  be  used,  or  a  multiple  thereof  for  any  cor- 
responding volume. 

8.  As  clarifying  and  decolorizing  reagents  there  may  be  used : 
(a)  subacetate  of  lead,  (3  parts  by  weight  of  acetate  of  lead,  one 
part  by  weight  of  oxide  of  lead,  10  parts  by  weight  of  water) ;  (b] 
alumina  cream  ;  (c)  concentrated  solution  of  alum.     Boneblack  and 
decolorizing  agents  are  to  be  excluded. 

9.  After  bringing  the  solution  exactly  to  the  mark,  at  the  proper 
temperature,  and  after  wiping  out  the  neck  of  the  flask  with  filter 
paper,  all  of  the  well-shaken  clarified  sugar  solution  is  poured  upon 
a  dry  rapidly-acting  filter.     The  first  portions  of  the  filtrate  are  to 
be  rejected  and  the  rest,  which  must  be  perfectly  clear,  used  for 
polarization. 

Relation  of  Polarization  to  Percentage  of  Sucrose. 

In  the  absence  of  other  active  substances,  the  reading  of  the 
Ventzke  scale  as  obtained  above  shows  the  percentage  of  sucrose 
in  the  sample.  In  commercial  raw  sugar  the  only  other  sugars 
likely  to  be  present  are  dextrose  and  levulose.  If  the  total  amount 
of  these  reducing  sugars,  as  shown  by  Fehling's  solution,  is  small, 
the  value  of  the  sample  is  sufficiently  indicated  by  the  polarization, 
the  invert-sugar,  and  the  amounts  of  moisture  and  ash. 

Since,  however,  the  "  reducing  sugar "  contains  dextrose  and 
levulose  in  unknown  proportions,  the  determination  of  "  reducing 
power"  does  not  give  the  necessary  data  for  estimating  the  per- 
centage of  sucrose  in  the  sample.  The  percentage  of  sucrose  can, 
however,  be  determined  in  the  presence  of  a  known  mixture  of 
dextrose  and  levulose  by  observing  the  change  in  rotation  pro- 
duced by  hydrolysis.  This  is  unaffected  by  the  presence  of 
monosaccharides  since  the  latter  cannot  be  hydrolyzed. 

The  following  method  is  based  on  this  principle. 


CARBOHYDRATES— SPECIAL  METHODS.         91 

CLERGET'S  METHOD  FOR  SUCROSE. 

A  pure  sucrose  solution  polarizing  1 00°  on  the  Ventzke  scale  at 
a  temperature  of  20°,  will,  after  hydrolysis,  polarize  —  33.6°  on 
the  same  scale  at  the  same  temperature.  It  is  this  change  of  rota- 
tion from  right  to  left  which  gives  rise  to  the  terms  "  inversion  " 
for  hydrolysis,  and  "  invert-sugar  "  for  the  mixture  of  equal  parts  of 
dextrose  and  levulose  produced.  Since  the  laevorotary  power  of 
levulose,  and  hence  of  invert-sugar,  decreases  rapidly  with  increase 
of  temperature  the  reading  33.6°  would  be  found  only  at  20°. 
An  increase  of  1°  in  temperature  causes  a  decrease  of  0.5  degree 
Ventzke  in  the  laevorotation  of  the  invert-sugar,  and  at  about  87.2° 
the  reading  becomes  zero.  The  change  in  rotation  produced  by 
hydrolysis  in  pure  sucrose  solution  is,  therefore,  143.6  —  O.5/,  in 
which  t  is  the  temperature  at  which  the  readings  are  taken. 

To  estimate  the  sucrose  in  an  unknown  sample,  dissolve  26 
grams,  clarify,  dilute  to  100  c.c.,  filter  and  polarize  as  usual.  Place 
50  c.c.  of  the  filtrate  in  a  flask  marked  at  50  and  55  c.c.,  fill  to  the 
55  c.c.  mark  with  pure  concentrated  hydrochloric  acid  and  mix 
well.  Set  the  flask  in  water  and  heat  until  a  thermometer  sus- 
pended with  the  bulb  at  the  center  of  the  solution  reads  68°,  regu- 
lating the  heat  so  that  about  15  minutes  are  consumed  in  reaching 
the  required  temperature.  Remove  the  flask,  cool  quickly,  and 
polarize  in  a  220  mm.  tube  at  exactly  the  temperature  at  which  the 
first  reading  was  taken.  The  change  of  rotation  divided  by  that 
which  pure  sucrose  would  show  under  the  same  conditions,  gives 
the  percentage  of  sucrose  in  the  sample.  Or  stating  this  in  the 
form  of  an  equation 

a-b 


Sucrose  = 


143.6  —  0.5^ 


in  which  a  is  the  first  reading,  b  the  second  reading  and  /  the  tem- 
perature. 

Notes.  —  In  the  above  description  the  manipulation  of  Clerget 
and  the  figures  found  by  Borntrager  *  and  adopted  by  Wiley  f 
have  been  followed.  Landolt  adopts  other  conditions  requiring  a 
different  factor,  142.4  instead  of  143.6.  The  temperature  of  each 
polarization  must  be  carefully  noted.  Since  the  solution  is  increased 
one-tenth  in  volume  by  the  addition  of  hydrochloric  acid,  the  sec- 

*  Ztschr.   Vereins  Rubenzucker  Ind.,  1890,  876. 
f  Agricultural  Analysis,  Volume  III.,   108. 


'£ 
ot 


92  ORGANIC   ANALYSIS. 

ond  reading  must  be  increased  one  tenth  or  made  in  a  tube  one 
tenth  longer  than  that  first  used. 

The  Clerget  principle  can  be  applied  to  the  determination  of 
sucrose  in  the  presence  of  any  monosaccharide  or  mixture  of  mon- 
osaccharides.  By  the  use  of  citric,  in  place  of  hydrochloric,  acid 
it  can  also  be  used  for  mixtures  containing  lactose. 

DETERMINATION  OF  REDUCING  SUGAR. 

Usually  the  reducing  power  of  the  raw  sugar  is  determined  vol- 
umetrically,  as  described  in  the  preceding  chapter,  the  results  being 
calculated  as  percentage  of  invert-sugar.  For  the  determination 
dissolve  5  grams  of  the  sample  if  dark,  or  10  grams  if  light, 
dilute  to  100  c.c.  and  filter  through  dry  paper.  If  the  preliminary 
test  shows  the  solution  to  be  too  strong  or  too  weak,  prepare  a 
fresh  solution  for  the  final  determination.  When  the  volumetric 
method  is  carefully  carried  out,  the  results  are  sufficiently  exact  for 
all  ordinary  work.*  For  the  determination  of  very  small  amounts 
of  invert-sugar  the  method  introduced  by  Herzfeld,t  and  recom- 
mended by  the  International  Commission,  J  is  probably  the  most 
accurate. 

Herzfelds  Gravimetric  Method. 

Dissolve  20  grams  of  sample  in  80  c.c.  of  water,  clarify  with 
basic  lead  acetate  and  precipitate  the  excess  of  lead  by  sodium 
carbonate  §  (avoiding  more  than  a  slight  excess)  or  by  neutral 
potassium  oxalate  ;||  dilute  to  100  c.c.,  mix  thoroughly  and  filter 
through  dry  paper.  The  filtrate  must  be  perfectly  clear.  In  a 
beaker  of  250  c.c.  capacity,  place  50  c.c.  of  the  undiluted  "mixed 
Fehling  reagent"  (see  Fehling's  volumetric  method)  and  50  c.c.  of 
the  clarified  sugar  solution  ;  heat  at  such  a  rate  that  the  mixture 
boils  in  about  four  minutes,  and  boil  for  exactly  two  minutes* 
Add  TOO  c.c.  of  cold,  recently  boiled,  distilled  water,  filter  immedi- 
ately, and  find  the  amount  of  copper  reduced  as  described  under 
Defren's  method  in  the  preceding  chapter.  The  following  table 
shows  the  corresponding  percentage  of  invert-sugar. 

*  If  a  gravimetric  method  is  preferred,  use  that  of  Meissl  and  Hiller,  as  adopted  by 
the  Official  Agricultural  Chemists:   Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 
•\Ztschr.   Vereins  Rubenzucker  Industrie,  1 886,  6. 
\Journ.  Amer.  Chem.  Soc.,  1901,  23,  64. 
\  Bui.  46,  loc.  ctt.,  p.  33. 
||   Sawyer  :  Journ.  Amer.  Chem.  Soc.,  1904,  26,  1631. 


CARBOHYDRATES— SPECIAL  METHODS.         93 

HERZFELD'S  TABLE  FOR  THE  DETERMINATION  OF  INVERT  SUGAR  IN  MATERIALS 

CONTAINING  i  PER  CENT.  OR  LESS  OF  INVERT  SUGAR  AND  A 

HIGH  PERCENTAGE  OF  SUCROSE. 


1 
Copper    reduced 
by  10  grams 
of  material. 

Invert  sugar. 
Per  cent. 

Copper  reduced 
by  10  grams 
of  material. 

Invert  sugar. 
Per  cent. 

Copper  reduced 
by  10  grams 
of  material. 

Invert  sugar. 
Per  cent. 

Milligrams 

Milligrams. 

Milligrams. 

50 

0.05                        120 

0.40 

I90 

0.79 

55 

0.07                        125 

o.43 

195 

0.82 

60 
65 

O.OQ                        130 

o.ii                   135 

0-45 
0.48 

200 
205 

0.85 

0.88 

70 

O.I4                         140 

0.51 

2IO 

0.90 

75 

o.  16                   145 

0-53 

215 

0-93 

80 

0.19                   150 

0.56 

220                         0.96 

85 

0.21                           155 

o-59 

225                         0.99 

9° 

O.24 

1  60 

0.62 

230                         I.  O2 

95 

0.27 

I65 

0.65 

235 

1.05 

IOO 

0.30 

170 

0.68 

240 

1.07 

105 

0.32 

175 

0.71                  245                 i.io 

no 

0-35 

1  80 

0.74 

115 

0.38 

185 

0.76         II                       | 

DETERMINATION  OF  MOISTURE  AND  ASH. 

Dry  about  2  grams  of  sample  in  a  flat-bottomed  platinum  dish 
in  the  boiling  water  oven  until  the  loss  in  weight  on  heating  for 
one  hour  does  not  exceed  o.io  per  cent.  A  perfectly  constant 
weight  cannot  be  expected  if  invert-sugar  is  present,  since  levulose 
is  slowly  decomposed  by  heating  at  100°  in  the  air.  Calculate  the 
loss  of  weight  as  moisture. 

Moisten  the  residue  with  a  few  drops  of  concentrated  sulphuric 
acid  and  burn  to  whiteness.  Weigh;  and  report  the  result  as 
"sulphated  ash."  The  true  ash  is  considered  by  some  as  nine- 
tenths  and  by  others  as  four-fifths  of  the  "sulphated  ash." 


OFFICIAL  METHODS  AND  STANDARDS  OF  PURITY. 

Methods  of  the  Association  of  Official  Agricultural  Chemists  : 
Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

Regulations  governing  the  sampling  and  classification  of  im- 
ported sugars  and  molasses  :  Treasury  Department  Document  No. 
2113. 

German  official  methods :  Ztschr.  Untets.  Nahrungs-  und  Genus- 
smittel,  1903,  6,  1059;  Ztschr.  anal.  Chem.,  1903,  42  (Appendix  to 
Nos.  9  and  10). 

An  act  of  Congress  approved  June  3,  1902,  authorized  the  Sec- 
retary of  Agriculture  to  establish  standards  of  purity  for  food 


94  ORGANIC   ANALYSIS. 

products.     The  definitions  and  standards  established  for  sugar  and 
sugar  products  are  as  follows :  * 

1.  Sugar  is  the  product  chemically  known  as  sucrose  (saccha- 
rose)   chiefly  obtained    from    sugar    cane,  sugar  beets,  sorghum, 
maple,  or  palm. 

Standard  sugar  is  white  sugar  containing  at  least  ninety- nine 
and  five-tenths  (99.5)  per  cent,  of  sucrose. 

2.  Granulated,  loaf,  cut,  milled,  and  powdered  sugars  are  different 
forms  of  standard  sugars. 

3.  Maple  sugar  is  the  solid  product  resulting  from  the  evapora- 
tion of  maple  sap. 

4.  Massecuite,  melada,  mush   sugar,  and  concrete   are   products 
obtained  by  evaporating  the  purified  juice  of  a  sugar-producing 
plant,  or  a  solution  of  sugar,  to  a  solid  or  semi-solid  consistence  in 
which  the  sugar  chiefly  exists  in  a  crystalline  state. 

5.  Molasses  is  the  product  left  after  separating  the  sugar  from 
massecuite,  melada,  mush  sugar,  or  concrete. 

Standard  molasses  is  molasses  containing  not  more  than  twenty- 
five  (25)  per  cent,  of  water  nor  more  than  five  (5)  per  cent,  of  ash. 

6.  Sirup  is  the  product  obtained   by  purifying  and  evaporating 
the  juice  of  a  sugar-producing  plant  without  removing  any  of  the 
sugar. 

7.  Sugar-cane  sirup  is  a  sirup  obtained  by  the  evaporation  of 
the  juice   of  the   sugar   cane   or  by   the   solution  of  sugar-cane 
concrete. 

8.  Sorghum  sirup  is  a  sirup  obtained   by   the   evaporation    of 
sorghum  juice  or  by  the  solution  of  sorghum  concrete. 

9.  Maple  sirup  is  a  sirup  obtained  by  the  evaporation  of  maple 
sap  or  by  the  solution  of  maple  concrete. 

10.  Sugar  sirup  is  a  product  obtained  by  dissolving  sugar  to  the 
consistence  of  a  sirup. 

Standard  sirup  is  a  sirup  containing  not  more  than  thirty  (30) 
per  cent,  of  water  nor  more  than  two  and  five-tenths  (2.5)  per  cent. 
of  ash. 

DETERMINATION  OF  SUCROSE  IN  BEETS  AND  CANE. 
Sucrose  is  -the  only  sugar  found  in  appreciable  quantity  in  the 
fresh  juice  of  healthy  sugar  beets.  Sugar  cane  juice  contains  a 
small  amount  of  reducing  sugar  which  is  usually  considered  to  be 
invert-sugar,  but  which  according  to  Wiley  f  is  without  influence 
upon  polarized  light.  In  either  case  therefore  the  polarization  of 

*  U.  S.  Dept.  of  Agriculture,  Office  of  the  Secretary,  Circular  No.  10  (November, 
1903).  See  also  Circular  No.  13. 

f  Agricultural  Analysis,  Volume  III.,  p.  234..  The  reducing  substances  of  the 
original  cane  juice  should  not  be  confused  with  the  invert-sugar  produced  by  hydro- 
lysis of  sucrose,  in  the  process  of  sugar  manufacture. 


CARBOHYDRATES— SPECIAL  METHODS.         95 

a  properly  clarified  sample  is  considered  as  showing  the  amount 
of  sucrose  present. 

Beets  after  being  thoroughly  cleaned  (the  loss  of  weight  in 
cleaning  being  determined  and  reported  as  "tare")  are  reduced  to 
a  fine  pulp  by  rasping  or  grinding.  This  pulp  is  then  treated  in 
one  of  three  ways  (i)  separating  the  insoluble  matter  by  pressure 
and  polarizing  the  juice,  (2)  extracting  with  alcohol,  (3)  extracting 
with  water.  The  first  method  introduces  errors  from  imperfect 
separation  of  the  juice,  the  second  is  longer  and  more  expensive 
than  the  third  which  is  now  generally  used.  If,  however,  the 
pulp  be  treated  with  water  and  filled  to  volume  in  a  graduated 
flask  as  in  the  analysis  of  raw  sugar  it  is  very  difficult  to  expel  the 
air  bubbles  entirely  from  the  mixture  of  pulp  and  water.  The 
following  method  described  by  Sachs  *  avoids  this  inconvenience 
and  has  been  found  satisfactory  for  rapid  work  :  Weigh  26  grams 
of  the  pulp  in  a  beaker  and  add  177  c.c.  of  water  containing 
5  c.c.  of  the  usual  solution  of  basic  lead  acetate ;  shake  or  stir 
for  three  minutes  ;  filter  and  polarize  the  filtrate.  Twice  the  read- 
ing of  the  Ventzke  scale  is  approximately  the  percentage  of  suc- 
rose in  the  sample.  This  method  assumes  that  26  grams  of  beet 
pulp  contain  23  c.c.  of  juice,  making  the  total  volume  of  liquid  in 
the  mixture  200  c.c.  Many  German  sugar  chemists  hold  that 
extraction  with  water  gives  high  results  because  the  beet  contains 
substances  other  than  sucrose  which  are  soluble  in  water  and 
rotate  polarized  light  to  the  right.  According  to  Sachs,  however, 
these  substances  are  pectin-like  bodies  and  are  completely  preci- 
pitated by  basic  lead  acetate.  Trowbidgef  also  found  that  the 
results  obtained  by  digestion  in  water  were  practically  identical 
with  those  of  the  alcohol  extraction  method. 

In  sampling  sugar  cane  for  analysis,  the  canes  are  usually  cut 
into  thin  oblique  chips,  "cosettes,"  by  means  of  rotating  knives. 
These  cosettes  may  be  further  reduced  by  passing  through  a 
sausage  cutter  or  a  special  shredding  machine.  It  is  not  feasible, 
however,  to  reduce  the  cane  to  as  fine  a  pulp  as  is  obtained  from 
sugar  beets.  For  this  reason  more  time  and  a  higher  temperature 
must  be  used  to  ensure  complete  extraction  of  the  sucrose.  Ac- 
cording to  Wiley,  J  26  grams  of  the  chips  of  pulp  are  weighed  into  a 

*  Paper  read  before  the  Second  International  Congress  of  Applied  Chemistry,  Paris, 
1896;  quoted  by  Hiltner  and  Thatcher,  Journ.  Amer.  Chem.  Soc.,  1901,  23,  300. 
f  Journ.  Amer,  Chem.  Soc.,  1901,  23,  222. 
J  Agricultural  Analysis,  Vol.  HI.,  p.  238. 


96  ORGANIC  ANALYSIS. 

flask  graduated  at  102.6  c.c.,  this  graduation  being  based  on  the 
assumption  that  the  cane  contains  ten  per  cent,  of  insoluble 
matter  of  a  specific  gravity  about  equal  to  that  of  the  solution. 
The  flask  is  nearly  filled  with  water,  and  warmed  on  a  water-bath 
for  one  hour  with  frequent  shaking ;  then  filled  a  little  above  the 
mark  and  warmed  for  ten  minutes  longer  with  frequent  shaking. 
The  flask  is  now  cooled,  the  volume  adjusted  if  necessary  and 
the  liquid  filtered  through  dry  paper.  Fifty  c.c.  of  filtrate  are  col- 
lected in  a  55c.c.  flask,  clarified  with  basic  acetate,  filled  to  the 
mark,  shaken,  filtered,  and  the  filtrate  polarized  in  a  220  mm.  tube. 

For  further  information  on  the  analysis  of  sugar  beets  and  sugar 
cane,  the  following  books  and  papers  may  be  consulted: 

Wiley:  Agricultural  Analysis,  Vol.  III.,  Part  III. 

Spencer :  Handbook  for  Beet  Sugar  Chemists.  Handbook  for 
Sugar  Manufacturers. 

Lippmann  and  Pulvermacher :  Lunge's  Chemisch-technische 
Untersuchungsmethoden. 

Friihling  and  Schultz:  Anleitung  zur  Untersuchung  fiir  die 
Zuckerindustrie. 

Trowbridge :  Notes  on  Sugar  Beets,  Journ.  Amer.  Chem.  Soc., 
1901,  23,  216. 

Hiltner  and  Thatcher:  An  Improved  Method  for  the  Rapid 
Estimation  of  Sugar  in  Beets,  Journ.  Amer.  Chem.  Soc.,  1901,  23, 
299,  863. 

COMMERCIAL  GLUCOSE. 
OFFICIAL  DEFINITIONS  AND  STANDARDS  OF  PURITY.  * 

i.  Starch  sugar  or  grape  sugar  is  the  solid  product  obtained  by 
hydrolyzing  starch  or  a  starch-containing  substance  until  the 
greater  part  of  the  starch  is  converted  into  dextrose.  Starch  sugar 
or  grape  sugar  appears  in  commerce  in  two  forms,  anhydrous  and 
hydrous.  In  the  former  the  sugar  is  crystallized  without,  in  the 
latter  with,  water  of  crystallization.  The  hydrous  varieties  are 
commonly  known  as  70  and  80  sugars ;  70  sugar  is  also  known  as 
brewers'  sugar,  and  80  sugar  as  climax  or  acme  sugar. 

(a)  Standatd  70  sugar  or  brewers  sugar  is  hydrous  starch  sugar 
containing  not  less  than  70  per  cent,  of  dextrose  and  not  more  than 
O.8  per  cent,  of  ash. 

*U.  S.  Dept.  Agriculture,  Office  of  the  Secretary,  Circulars  10  and  13. 


CARBOHYDRATES— SPECIAL  METHODS.        97 

(&)  Standard  80  sugar,  climax  ot  acme  sugar,  is  hydrous  starch 
sugar  containing  not  less  than  80  per  cent,  of  dextrose  and  not 
more  than  1.5  per  cent,  of  ash. 

(c]  Standard  anhydrous  grape  sugar  is  anhydrous  grape  sugar 
containing  not  less  than  95  per  cent,  of  dextrose  without  water  of 
crystallization  and  not  more  than  O.8  per  cent,  of  ash.  • 

The  ash  of  these  standard  products  consists  almost  entirely  of 
chlorides  and  sulphates. 

2.  Glucose,  mixing  glucose,  or  confectioners'  glucose  is  a   thick 
sirupy  substance  obtained  by  incompletely  hydrolyzing  starch   or 
a  starch-containing  substance,  decolorizing   and   evaporating  the 
product. 

Standard  glucose,  mixing  glucose,  confectioners  glucose  is  colorless 
glucose,  vaiying  in  density  between  41  and  45  degrees  Baume,  at 
a  temperature  of  100  degrees  F.  It  conforms  in  density,  within 
these  limits,  to  the  degree  Baume  it  is  claimed  to  show,  and  for  a 
density  of  41  degrees  Baume  contains  not  more  than  21  per  cent, 
of  water,  and  for  a  density  of  45  degrees  not  more  than  14  per  cent. 
It  contains  on  a  basis  of  41  degrees  Baume  not  more  than  I  per 
cent,  of  ash,  consisting  chiefly  of  chlorides  and  sulphates. 

3.  Glucose  sirup  or  corn  sirup  is  glucose  unmixed  or  mixed  with 
sirup,  molasses,  or  refiners  sirup  [the  latter  being  defined  in  Circu- 
lar No.  13  as  the  residual  liquid  product  ("treacle")  obtained  in 
the  process  of  refining  raw  sugars]. 

Standard  glucose  sirup  or  corn  sirup  is  glucose  sirup  or  corn  sirup 
containing  not  more  than  25  per  cent,  of  water  nor  more  than  3 
per  cent,  of  ash. 

ANALYSIS  BY  WILEY'S  METHOD.* 

This  method  was  devised  for  the  analysis  of  mixtures  resulting 
from  incomplete  hydrolysis  of  starch,  and  is  based  on  the  assump- 
tion that  dextrose  and  maltose  are  oxidized  to  optically  inactive 
products  when  heated  with  an  alkaline  solution  of  mercuric  cya- 
nide and  that  dextrin  is  unaffected  by  the  treatment  and  shows  a 
specific  rotation  of  +  193. 

Dissolve  10  grams  of  sample  in  60  to  70  c.c.  of  water  in  a  100 
c.c.  flask;  boil  for  10  to  15  minutes  to  secure  a  normal  rotation  of 
the  reducing  sugars;  cool,  add  some  alumina  cream,  if  the  solution 

*  Ghent.  News,  1882,  46,  175  ;  Agricultural  Analysis,  Vol.  III.,  pp.  286-293. 


98  ORGANIC   ANALYSIS. 

is  not  clear,  and  fill  to  the  mark.  After  thorough  mixing,  filter 
and  polarize  in  a  200  mm.  tube.  Calculate  the  result  as  apparent 
specific  rotation  of  the  original  sample. 

To  10  c.c.  of  the  same  filtrate  add  50  c.c.  of  the  cyanide  solu- 
tion* and  boil  for  3  to  5  minutes.  Add  hydrochloric  acid  to  faint 
excess,  when  the  brown  color  of  the  solution  will  disappear;  cool, 
and  dilute  the  solution  to  100  c.c.  Filter  (through  animal  char- 
coal if  necessary)  and  polarize  in  the  same  instrument  as  before. 
This  result  is  calculated  to  apparent  specific  rotation  as  before, 
basing  the  calculation  on  the  entire  amount  of  the  original  sub- 
stance. 

Dilute  10  c.c.  of  the  filtrate  first  obtained  to  100  c.c.  and  deter- 
mine the  reducing  power  by  Defren's  method  (preceding  chapter), 
or  by  Allihn's  method,  as  given  under  determination  of  starch. 
Calculate  the  reducing  power  as  percentage  of  dextrose  in  the 
original  sample. 

Let  P=  the  first  polarization   calculated  as  apparent   specific 

rotation  (due  to  dextrose,  maltose  and  dextrin). 
Pf  =  the  second  polarization  calculated  as  apparent  specific 

rotation  (due  to  dextrin). 

R  =  the  reducing  power  calculated  as  dextrose. 
d  =  dextrose. 
m  =  maltose. 
d'  =  dextrin. 

Assuming  the  reducing  power  of  maltose  to  be  0.61  that  of 
dextrose,  and  the  value  \_OL]D  to  be  respectively,  dextrose,  53; 
maltose,  138;  dextrin,  193: 


P'  =  ig$dr. 
R  —  d  +  o.6im. 

Solve  for  d,   m  and  d'   to   obtain  the  amounts  of   dextrose, 
maltose  and  dextrin  in  the  original  sample. 

ANALYSIS  BY  OTHER  METHODS. 

Several  other  methods  for  the  analysis  of  commercial  glucose 
have  been  proposed.     Some  of  these  do  not  provide  for  the  esti- 

*  Prepared  by  dissolving  120  grams  of  mercuric  cyanide  in  water,  adding  1  20  grams 
of  sodium  hydroxide  and  diluting  the  solution  to  I  liter. 


CARBOHYDRATES— SPECIAL  METHODS.         99 

mation  of  maltose,  but  attribute  the  entire  reducing  power  to  dex- 
trose. Others,  including  that  of  Allen,  assume  that  dextrin,  mal- 
tose and  dextrose  are  the  only  organic  compounds  present.  The 
percentage  of  solids  is  usually  estimated  from  the  specific  gravity, 
the  ash  determined  and  deducted.  Assuming  that  the  total  per- 
centage of  dextrose,  maltose  and  dextrin  is  now  known,  the 
amount  of  each  can  be  found  by  one  determination  of  the  appa- 
rent specific  rotation  and  a  determination  of  the  reducing  power. 
For  details  of  the  methods  consult  Allen's  Commercial  Organic 
Analysis,  Volume  I.  (third  edition),  pages  358-378,  and  the  fol- 
lowing papers : 

Brown,  Morris  and  Millar:  Experimental  Methods  Employed 
in  the  Examination  of  the  Products  of  Starch  Hydrolysis,  Journ. 
C/iem.  Soc.,  1897,  71*  72- 

Rolfe  and  Defren  :  An  Analytical  Investigation  of  the  Hydroly- 
sis of  Starch  by  Acid,Joum.  Amer.  Chem.  Soc.,  1896,  18,  869. 

Rolfe  and  Faxton :  The  Exact  Estimation  of  Total  Carbohy- 
drates in  Acid  Hydrolized  Starch  Products,  Journ.  Amer.  Chem.  Soc.t 
1897,  19,  698. 

Honig :  Ueber  Zusammensetzung  und  Untersuchung  von  Star- 
kesyrupen,  Ztschr.  Llnters.  Nahr.-Genussm.,  1902,  5,  641. 


CHAPTER  VII. 
Carbohydrates — Special  Methods. 

( Continued. ) 
THE  DETERMINATION  OF  STARCH. 

METHOD  OF  DIRECT  ACID  HYDROLYSIS. 

This  method  is  accurate  for  the  determination  of  starch  in  such 
samples  as  contain  no  other  substance  insoluble  in  water  and  cap- 
able of  yielding  reducing  sugar  on  heating  with  dilute  acid.  It  is 
now  well  known  that  such  interfering  substances,  especially  the 
pentosans,  are  generally  found  in  vegetable  tissues,  and  that  the 
results  obtained  by  direct  acid  hydrolysis  are  usually  too  high. 
This  is,  however,  the  method  which  has  been  commonly  used  until 
recently  and  for  comparisons  with  earlier  work  determinations  by 
direct  hydrolysis  are  still  frequently  required. 

Conversion  of  StarcJi  to  Dextrose* 

Weigh  2  to  5  grams  and,  if  much  fat  is  present,  wash  with  four 
or  five  successive  portions  each  10  c.c.  of  ether;  allow  the  ether  to 
evaporate,  and  wash  with  150  c.c.  of  10  percent,  alcohol  tore- 
move  soluble  carbohydrates.  Wash  the  residue  into  a  250  c.c. 
flask  with  200  c.c.  of  water,  add  20  c.c.  of  hydrochloric  acid  of 
1.125  sp.  gr.,  and  heat  in  a  boiling  water  bath  with  a  reflux  con- 
denser for  two  and  one-half  hours.  Cool  to  room  temperature, 
nearly  neutralize  with  sodium  hydroxide,  dilute  to  250  c.c. ;  filter, 
and  determine  dextrose  in  a  portion  of  the  filtrate,  using  either 
Defren's  method,  already  described,  or  Allihn's  method,  as  follows  : 

Allihris  Method  for  the  Determination  of Dextrose. ,| 
Reagents.  —  (i)  34.64  grams  of  crystallized  copper  sulphate  dis- 
solved in  water  and  diluted  to  500  c.c. 

(2)  173  grams  of  sodium  potassium  tartrate  and  125  grams  of 
potassium  hydroxide  dissolved  in  water  and  diluted  to  500  c.c. 

*Sachsse:  CAem.  CentrbL,  1877,  732;  Bui.  46,  Bur.  Chem.  U.  S.  Dept.  Agri- 
culture. 

•\Journ.  prakt.  Chem.,  1880,  22,  46  ;  Bui.  46,  he.  cit. 


CARBOHYDRATES— SPECIAL    METHODS.        101 

Determination.  —  Place  30  c.c.  of  the  copper  solution,  30  c.c.  of 
the  alkaline  tartrate  solution  and  60  c.c.  of  water  in  a  beaker  or 
casserole  and  heat  to  boiling.  To  the  boiling  liquid  add  25  c.c. 
of  the  dextrose  solution,  note  the  time  at  which  the  mixture  begins 
to  boil  and  continue  the  boiling  for  exactly  two  minutes.  Filter  at 
once  and  obtain  the  weight  of  copper  in  the  precipitated  cuprous 
oxide  by  any  of  the  methods  suggested  in  connection  with  Defren's 
process  described  in  Chapter  V,  or  as  follows:  Collect  the 
cuprous  oxide  on  an  asbestos  filter  in  a  Gooch  crucible;  wash 
the  precipitate  (including  any  which  may  adhere  to  the  beaker  and 
which  need  not  be  transferred  to  the  filter)  thoroughly  with  hot 
water,  transfer  the  asbestos  and  adhering  oxide  from  the  crucible 
to  the  beaker.  Dissolve  the  oxide  still  remaining  in  the  crucible 
by  means  of  I  to  2  c.c.  of  concentrated  nitric  acid,  adding  the  acid 
from  a  pipette  and  receiving  the  solution  in  the  beaker  containing 
the  asbestos  and  the  main  part  of  the  precipitate.  Rinse  the 
crucible  with  a  jet  of  water  allowing  the  rinsings  to  flow  into  the 
beaker.  Heat  the  contents  of  the  beaker  until  all  copper  is  in 
solution;  filter,  wash  thoroughly,  dilute  the  filtrate  to  100  to  150 
c.c.,  add  one  drop  of  concentrated  sulphuric  acid  and  determine 
copper  by  electrolysis.  Find  the  corresponding  weight  of  dextrose 
from  Allihn's  table. 

Notes.  —  The  conditions  described  must  be  observed  carefully. 
The  dextrose  solution  added  to  the  copper  reagent  must  be  free 
from  turbidity  and  only  faintly  acid.  The  rapid  addition  of  this 
cold  solution  stops  the  boiling  of  the  reagent,  and  it  is  well  to  have 
the  heat  so  regulated  that  the  mixture  will  boil  again  in  about 
two  minutes,  then  after  exactly  two  minutes  of  actual  boiling  re- 
move the  flame  and  filter  the  solution  at  once.  The  cuprous  oxide 
is  very  apt  to  run  through  the  filter.  To  prevent  this,  after  making 
the  asbestos  filter  as  usual,  pour  on  it  some  very  fine  asbestos 
suspended  in  water,  so  as  to  form  a  tight  layer  on  the  top  of 
the  felt.  On  the  assumption  that  the  starch  is  quantitatively 
hydrolyzed  to  dextrose,  the  weight  of  the  latter  multiplied  by  0.9 
gives  the  corresponding  weight  of  starch.  For  detailed  studies  of 
the  hydrolysis  of  starch  by  acids,  see  the  papers  of  Rolfe*  and  of 
Noyes.t 


*Journ.  Amer.  Chem.  Soc.y  1896,  18,  869;   1897,  19,  261  ;   1903,  25,  1003,  1015 
-\  Ibid.,  1904,  26,  266. 


102 


ORGANIC   ANALYSIS. 


ALLIHN'S  TABLE  FOR  THE  DETERMINATION  OF  DEXTROSE. 


Milli- 
grams 
of 
Copper. 

Milli- 
grams 
of  Dex- 
trose. 

Milli- 
grams 
of 
Copper. 

Milli- 
grams 
of  Dex- 
trose. 

Milli- 
grams 
of 
Copper. 

Milli- 
grams 
of  Dex- 
trose. 

Milli- 
grams 
of 
Copper. 

Milli- 

of  Dex- 
trose. 

Milli- 
grams 
of 
Copper. 

Milli- 
grams 
of  Dex- 
trose. 

10 

6.1 

67 

34-3 

I24 

63.1 

181 

92.6 

238 

122.8 

II 

6.6 

68 

34-8 

125 

63.7 

182 

93-1 

239 

123.4 

12 

7-i 

69 

35-3 

126 

64.2 

183 

93-7 

240 

123.9 

J3 

7-6 

70 

35-8 

127 

64.7 

184 

94-2 

24!          124.4 

14 

8.1 

71 

36.3 

128 

65.2 

185 

94-7 

242 

125.0 

15 

8.6 

72 

36.8 

I29 

65-7 

180 

95-2 

243 

125-5 

16 

9.0 

73 

37-3 

130 

66.2 

187 

95-7 

244 

I26.O 

17 

9-5 

74 

37-8 

!3I 

66.7 

1  88 

96.3 

245 

126.6 

18 

IO.O 

75 

38.3 

I32 

67.2 

189 

96.8 

246 

127.1 

19 

10.5 

76 

38.8 

133 

67.7 

190 

97-3 

247 

127.6 

20 

II.  0 

77 

39-3 

134 

68.2 

191 

97-8 

248 

I28.I 

21 

"•S 

78 

39-8 

135 

68  8 

192 

98.4 

249         128.7 

22 

12.0 

79 

40-3 

136 

69.3 

193 

98.9 

250 

129.2 

23 

12-5 

80 

40.8 

137 

69.8 

194 

99-4 

25  * 

129.7 

24 

13.0 

81 

41-3 

138 

70.3 

^95 

1  00.0 

252 

130.3. 

25 

13-5 

82 

41.8 

139 

70.8 

196 

100.5 

253 

130.8 

26 

14.0 

83 

42.3 

I4O 

7*-3 

197 

IOI.O 

254 

I3I-4 

27 

14-5 

84 

42.8 

I4I 

71.8 

198 

101.5 

255 

W-9 

28 

15-0 

85 

43.4 

142 

72.3 

199 

IO2.O 

256 

132.4 

29 

'5-5 

86 

43-9 

H3 

72.9 

200 

102.6 

257 

133-0 

3° 

16.0 

87 

44.4 

144 

73-4 

201 

I03.I 

258 

133-5 

31 

16.5 

88 

44-9 

145 

73-9 

202 

I03-7 

259 

I34-I 

32 

17.0 

89 

45-4 

146 

74-4 

203 

104.2 

260 

134.6 

33 

17-5 

90 

45-9 

147 

74-9 

204 

104.7 

26l 

I35-I 

34 

18.0 

9i 

46.4 

148 

75-5 

205 

105-3 

262 

135-7 

35 

18.5 

92 

46.9 

149 

76.0 

206 

105.8 

263 

136.2 

36 

18.9 

93 

47-4 

150 

76.5 

207 

106.3 

264 

136.8 

37 

19.4 

94 

47-9 

151 

77.0 

208 

106.8 

265          137.3 

38 

19.9 

95 

48.4 

152 

77-5 

2O9 

107.4 

266          I37.8 

39 

20.4 

96 

48.9 

«S3 

78.1 

2IO 

107.9 

267      i     138.4 

40 

20.9 

97 

49-4 

i54 

78.6 

211 

108.4 

268          138.9 

41 

21.4 

98 

49-9 

i55 

79.1 

212 

109.0 

269    :    139-5 

42 

21.9 

99 

50-4 

156 

79.6 

213 

109.5 

270    i    140.0 

43 

22.4 

100 

50-9 

i57 

80.  i 

214 

I  IO.O 

271     |    140.6 

44 

22.9 

101 

5«-4 

158 

80.7 

215 

1  10.6 

272 

141.1 

45 

23-4 

102 

5i.9 

i59 

81.2 

216 

in.  i 

273       Hi-? 

46 

23-9 

103 

52.4 

1  60 

81.7 

217 

in.  6 

274       142.2 

47 

24.4 

104 

52.9 

161 

82.2 

218 

112.  1 

275 

142.8 

48 

24.9 

!05 

53-5 

162 

82.7 

219 

112.7 

276 

143-3 

49 

25-4 

106 

54-0 

163 

83-3 

22O 

113.2 

277       143-9 

5° 

25.9 

107 

54-5 

164 

83.8 

221 

"3-7 

278       144.4 

51 

26.4 

1  08 

55-0 

165 

84-3 

222 

114-3 

279       145-0 

52 

26.9 

109 

55-5 

166 

84.8 

223 

114.8 

280       145.5 

53 

27.4 

no 

56.0 

167 

85.3 

224 

"5-3 

281       146.1 

54 

27.9 

III 

56.5 

1  68 

85-9 

225 

II5-9 

282       146.6 

55 

28.4 

112 

57-0 

169 

86.4 

226 

116.4 

283    j   147-2 

56 

28.8 

"3 

57-5 

170 

86.9 

227          116.9 

284    |    147.7 

57 

29-3 

114 

58.0 

171 

87-4 

228 

117.4 

285        148.3 

58 

29.8 

"5 

58.6 

172 

87-9 

229 

118.0 

286.     ;     148.8 

59 

30-3 

116 

59-1 

173 

88.5 

230 

118.5 

287"         149.4 

60 

30.8 

117 

59-6 

174 

89.0 

231 

119.0 

288         149.9 

61 

3'-3 

118 

60.  i 

175 

89.5 

232 

119.6 

299         150.5 

62 

31.8 

119 

60.6 

176 

90.0 

233 

1  20.  i 

290         I5I.O 

63 

32.3 

1  20 

6l.i 

177 

90-5 

234 

120.7 

291          I5I.6 

64 

32.8 

121 

61.6 

178 

91.1 

235 

121.  2 

292          I52.I 

65 

33-3 

122 

62.1 

179 

91.6 

236 

I2I.7 

293 

152.7 

66 

33-8 

123             62.6 

1  80 

92.1 

237 

122.3 

294 

153-2 

CARBOHYDRATES— SPECIAL    METHODS.         103 

ALLIHN'S  TABLE  FOR  THE  DETERMINATION  OF  DEXTROSE. — Continued. 


Milli- 

Milli-         Milli- 

Milli- 

Milli- 

Miili-      ||    Milli- 

Milli- 

Milli- 

Milli- 

grams 
of 

grams         grams 
ofDex-            of 

grams 
of  Dex- 

.  grams 
of 

grams         grams 
of  Dex-    '  '       of 

grams 
of  Dex- 

grams 
of 

grams 
of  Dex- 

Copper. 

trose. 

Copper. 

|      trose. 

Copper. 

trose. 

Copper 

trose. 

Copper 

trose. 

295 

153.8 

329 

172.5 

363 

I9I.7 

397 

211.  2 

431 

231.0 

296 

!54-3 

330 

I73-I 

364 

I92.3 

398 

2II.7 

432 

231.6 

297 

154.9 

331 

173-7 

365 

I92.9 

399 

212.3 

433 

232.2 

298 

155.4 

332 

174.2 

366 

193-4 

400 

212.9 

434 

232.8 

299 

156.0 

333 

174.8 

367 

194.0  !!  401 

213-5 

435 

233.4 

300 

156-5 

334 

175-3 

368 

194.6 

402 

2I4.I 

436 

233.9 

3OI 

I57.I 

335 

!75-9 

369 

195-1 

403 

214.6 

437 

2345 

302 

157.6 

336 

176.5 

370 

195-7 

404 

215.2 

438 

235-1 

303 

158.2 

337 

177.0 

371 

196.3 

405 

215.8 

•  439 

235.7 

3°4 

158.7 

338 

177.6 

372 

196.8 

406 

216.4 

440 

236.3 

305 

159.3 

339 

178.1 

373 

197.4 

407 

217.0 

441 

236.9 

306 

159.8 

340 

178.7 

374 

198.0 

408 

217-5 

442 

237.5 

3°7 

160.4 

34i 

!79-3 

375 

198.6 

409 

2I8.I 

443 

238.1 

308 

160.9 

342 

179-8 

376 

199.1 

410 

218.7 

444 

238.7 

3°9 

161.5 

343        180.4 

377 

199.7 

411 

219.3 

445 

239.3 

310 

162.0 

344        180.9 

378 

200.3 

412 

219.9 

446 

239.8 

3" 

162.6 

345        181.5 

379 

200.8 

413 

220.4 

447 

240.4 

312 

163.1 

346 

182.1 

380 

201.4 

414 

221.0 

448 

241.0 

313 

163.7 

347 

182.6 

38i 

202.0 

415 

221.6 

449 

241.6 

3H 

164.2 

348 

183.2 

382 

202-5 

416 

222.2 

45° 

242.2 

315 

164.8 

349 

183.7 

383 

203.1 

417 

222.8 

45i 

242.8 

3i6 

165.3 

35° 

184.3 

384 

203.7 

418 

223.3 

452 

243-4 

317 

165.9 

35i 

184.9 

385 

204.3 

419 

223.9 

453 

244.0 

3i8 

166.4 

352 

185.4 

386 

204.8 

420 

224.5 

454 

244.6 

3!9 

167.0 

353 

186.0 

387 

205.4    ;!    421 

225.1 

455 

245.2 

320 

167.5 

354 

186.6 

388       206.0 

422 

225.7 

456 

245-7 

321 

168.1 

355 

187.2 

389 

206.5 

423 

226.3 

457 

246.3 

322 

168.6 

356 

187.7 

390 

207.1 

424 

226.9 

458 

246.9 

323 

169.2 

357 

188.3 

39i 

207.7 

425 

227.5 

459 

247-5 

324 

169.7 

358 

188.9 

392 

208.3 

426 

228.0 

460 

248.1 

325 

170.3 

359 

189.4 

393    i    208.8 

427 

228.6 

461 

248.7 

326 

170.9 

360 

190.0 

394 

209.4 

428 

229.2 

462 

249-3 

327 

171.4 

36i 

190.6 

395 

210.0 

429 

229.8 

463 

249.9 

328 

172.0 

362 

191.1 

396 

210.6     J 

430 

230.4     • 

According  to  several  investigators,*  the  weight  of  dextrose  should 
be  multiplied  by  a  higher  factor  (0.917  to  0.941)  rather  than  0.9,  to 
find  the  corresponding  weight  of  starch. 


METHOD  OF  DIGESTION  WITH  DIASTASE  OR  SALIVA. 
If  starch  is  gelatinized  by  boiling  with  water  and  then  treated 
with  malt  diastase  or  saliva,  it  can  be  converted  into  maltose  and 
dextrin  and  these  separated  by  filtration  from  the  insoluble  residue 

*  Salomon:  Ztschr.  anal.  Chem.,  1883,  22,  593.  Soxhlet  :  Wochenschr.  fur 
Brauer.,\$&$,  193;  Vaubel,  II.,  455.  Sostegni  :  Chem.  Centrbl.,  1887,  58,  896. 
Lintner  and  Dull  :  Ztschr.  angew.  Chem.,  1891,  537.  Ost :  Chem.  Ztg.,  1895,  19, 
1502.  Rcissing  :  Ztschr.  offentl.  Chem.,  1904,  10,  6l  ;  Abs.  Journ.  Chem.  Soc.,  1904, 
86,  ii,  298.  Noyes  :  Journ.  Amer.  Chem.  Soc.,  1904,  26,  280. 


104  ORGANIC   ANALYSIS. 

containing  the  pentosans  and  other  substances  which  cause  the 
results  by  the  preceding  method  to  be  too  high. 

Determination,  —  Extract  2.5  to  5  grams  of  sample  with  five  suc- 
cessive portions  each  10  c.c.  of  ether,  decanting  the  washings 
through  a  hardened  filter;  wash  with  150  c.c.  of  10  per  cent, 
alcohol;*  transfer  the  residue  to  a  beaker  with  50  to  100  c.c.  of 
water;  heat  gradually  to  boiling,  stirring  constantly  to  prevent 
bumping  or  the  formation  of  lumps.  Cool  to  55°  for  diastase,  or  to 
38°  for  saliva;  add  the  solution  containing  the  enzyme  and  keep 
the  mixture  within  two  degrees  of  the  stated  temperature  until  a 
drop,  removed  and  tested  on  a  porcelain  plate,  no  longer  shows  a 
reaction  for  starch  on  mixing  with  a  drop  of  dilute  solution  of  iodine 
in  aqueous  potassium  iodide.  Now  heat  the  solution  again  to  boil- 
ing in  order  to  gelatinize  any  starch  granules  which  may  remain ; 
test  the  solution,  and  if  starch  is  found,  cool  to  the  proper  tempera- 
ture ;  add  more  of  the  enzyme  and  digest  as  before.  Continue  this 
treatment  until  the  solution  gives  no  starch  reaction  after  boiling,  or 
until  a  careful  microscopic  examination  shows  that  the  insoluble 
residue  is  entirely  free  from  starch.  Dilute  to  250  c.c.,  mix  thor- 
oughly and  pour  on  a  dry  fluted  filter.  Transfer  150  c.c.  of  the 
filtrate  to  a  250  c.c.  flask ;  add  15  c.c.  of  hydrochloric  acid  of  1.125 
sp.  gr.,  attach  the  flask  to  a  reflux  condenser  and  heat  in  a  boiling 
water-bath  for  two  and  one-half  hours,  or  boil  gently  on  a  hot 
plate  or  sand-bath  for  35  to  45  minutes.  Complete  the  determi- 
nation as  described  in  the  preceding  method. 

The  determination  should  be  carried  through  without  interrup- 
tion. If  this  is  impossible,  care  must  be  taken  to  avoid  alcoholic 
or  lactic  fermentation.  After  the  digestion  with  the  enzyme  is  fin- 
ished, but  not  before,  salicylic  acid  may  be  added  as  a  preserva- 
tive. It  has  been  recommended  that  a  trace  of  fluoride  be  added 
at  the  start  to  retard  lactic  fermentation  while  the  digestion  with 
the  enzyme  is  taking  place. 

When  only  a  few  determinations  are  to  be  made,  freshly  col- 
lected saliva  can  conveniently  be  used,  as  this  is  free  from  carbo- 
hydrate. If  commercial  diastase  or  an  infusion  of  malt  f  is  used, 


*  This  extraction  can  often  be  omitted,  since  for  many  purposes  it  is  unnecessary  to 
distinguish  between  starch  and  soluble  carbohydrates. 

|  An  active  malt  infusion  can  be  prepared  by  digesting  10  grams  of  fresh,  finely- 
ground  malt,  over  night  at  room  temperature,  with  20 3  c.c.  of  water  or  10  per  cent, 
alcohol. 


CARBOHYDRATES— SPECIAL    METHODS. 


105 


the  amount  added  must  be  noted  and  a  correction  applied  for  the 
carbohydrate  thus  introduced.  This  is  found  by  heating  a  quantity 
of  the  diastate  or  infusion  with  acid  and  determining  the  resulting 
dextrose  as  in  the  starch  determination, 

COMPARISON  OF  RESULTS. 

The  diastase  method,  carefully  carried  out,  is  believed  to  yield 
practically  correct  results.  As  already  explained,  the  results  ob- 
tained by  direct  acid  hydrolysis  are  usually  higher  owing  to  the 
presence  of  other  substances  which  yield  reducing  sugars.  A 
comparison  of  the  results  of  the  two  methods  is  of  consider- 
able interest,  both  because  many  of  the  recorded  determinations 
of  starch  were  made  by  the  acid  method  and  because  the  deter- 
mination of  copper-reducing  substance  obtained  by  direct  hydrol- 
ysis is  sometimes  used  as  a  means  of  detecting  adulterants  in 
spices.  The  table  below  shows  the  results  of  comparison  of  the 
two  methods  on  a  variety  of  substances.  Many  of  the  results 
of  Winton  and  associates  are  averaged  from  the  comparative 
examination  of  several  samples.  The  other  results  were  obtained 
by  the  writer,  only  one  sample  of  each  kind  being  examined. 

Results  by  Diastase  and  by  Direct  Acid  Hydrolysis. 


Substance. 

Starch  Indicated  by 

Substance. 

Starch  Indicated  by 

Diastase 
Method 
Per  Cent. 

Acid 
Method 
Per  Cent. 

Diastase 
Method 
Per  Cent. 

Acid 
Method 
Per  Cent. 

Air-dry  starch  

82.49 

66.55 
56.23 
55-32 
20.97 
14.06 
8.07 

4-39 
4.14 
1.46 
0.96 

82.30 

68.35 
59-01 
58.63 
38.82 
21.15 
ii.  16 
22.69 
18.03 
20.51 
20.13 

White  pepper*  

56.47 
39-55 
34-15 
27.87 
23.72 

3.04 

2-74 
2.30 

1.  01 
1.  01 

0.84 

59-17 
42.88 
38.63 
31-73 
25-56 
18.03 
8.99 
H-43 

8-47 
19.30 
22.72 

Wheat  flour  

Oatmeal  .  

Black  pepper*  
Mace* 

Graham  flour  

Wheat  bran  

Nutmeg* 

Linseed  meal*  

Allspice* 

Cocoa  nibs*  

Cloves* 

Wheat  straw   .. 

Pepper  shells*  
Cayenne* 

Cocoa  shells*  

Buckwheat  hulls*.... 
Corn  stover  

Walnut  shells*  
Almond  shells*  

DETERMINATION  OF  STARCH  IN  MEAT  PRODUCTS. 
Starchy  materials  are  sometimes  added  as  "  fillers  "  to  sausages 
and  other  forms  of  chopped  meat.     This  adulteration  is  easily  de- 

*  Results  by  Winton  and  associates,  compiled  from  Leach's  Food  Inspection  and 

Analysis. 


io6  ORGANIC   ANALYSIS. 

tected  by  the  iodine  reaction  which,  however,  must  be  carefully 
interpreted  since  a  small  amount  of  starch  may  legitimately  be 
present  from  the  spices  added  in  the  manufacture.  The  quantita- 
tive determination  is  complicated  by  the  fact  that  meat  appears  to 
contain  some  substance  which  interferes  with  the  separation  of  the 
cuprous  oxide  reduced  in  applying  the  usual  method.  Advantage 
is  therefore  taken  of  the  insolubility  of  starch  in  alcoholic,  and  its 
solubility  in  aqueous  potassium  hydroxide.  The  method  of  Mayr- 
hofer  as  modified  by  Bigelow  and  adopted  by  the  Association  of 
Official  Agricultural  Chemists  is  as  follows :  * 

Treat  from  10  to  20  grams  of  the  sample  under  examination 
(depending  upon  the  amount  of  starch  indicated  by  the  iodine  reac- 
tion) in  a  porcelain  dish  or  casserole  with  50  c.c.  of  an  8  per  cent, 
solution  of  potassium  hydroxide  and  heat  the  mixture  on  the  water- 
bath  until  the  meat  is  entirely  dissolved.  Add  an  equal  volume 
of  95  per  cent,  alcohol,  mix  thoroughly,  filter  the  mixture  through 
an  asbestos  filter  and  wash  twice  with  a  hot  4  per  cent,  solution 
of  potassium  hydroxide  in  50  per  cent,  alcohol.  Then  wash 
with  50  per  cent,  alcohol  until  a  small  portion  of  the  filtrate 
does  not  become  turbid  upon  the  addition  of  acid.  Return  the 
precipitate  and  filter  to  the  original  vessel  and  dissolve  the  pre- 
cipitate with  the  aid  of  heat  in  60  c.c.  of  a  normal  solution  of  po- 
tassium hydroxide.  In  the  case  of  sausage  with  a  high  starch  con- 
tent a  somewhat  larger  volume  of  alkali  may  be  required.  Acidify 
the  filtrate  strongly  with  acetic  acid,  dilute  to  a  definite  volume, 
mix  thoroughly  by  shaking,  filter  through  a  fluted  paper,  and  pre- 
cipitate the  starch  from  an  aliquot  part  of  the  filtrate  by  means  of 
an  equal  volume  of  95  per  cent,  alcohol.  Transfer  the  precipitate 
to  a  weighed  filter,  wash  thoroughly  with  50  per  cent,  alcohol,  with 
absolute  alcohol,  and  finally  with  ether,  dry  to  a  constant  weight  at 
the  temperature  of  boiling  water  and  weigh. 

ADDITIONAL  REFERENCES. 
Wiley:  Agricultural  Analysis,  Vol.  III. 
Winton:  Journ.  Anal.  Appl.  Chem.,  1888,  2,  153. 
Hibbard:  Journ.  Amer.  Chem.  Soc.,  1895,  17,  64. 
Ost:  Chem.  Ztg.,  1895,  19,  1501. 
Stone:  Journ.  Amer.  Chem.  Soc.\  1894,  16,  726. 
Sherman:  Analyst,  1897,22,  19. 

*Bul.  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


CARBOHYDRATES— SPECIAL    METHODS.         107 

Wiley  and  Krug :  Journ.  Amer.  Chem.  Soc.,  1898,  20,  253,  266. 
Lintner  :  Ztschr.  angew.  Chem.,  1898,  725. 
Lindet :  Journ.  Pharm.  Chim.t  1901,  [6],  14,  397. 
Witte:  Ztschr.  Unters.  Nakr.-Genussm.,  1904,  7,  65. 


SEPARATION   OF   CARBOHYDRATES    IN    CEREAL   PRODUCTS. 

DETERMINATION  OF  REDUCING  SUGARS,  SUCROSE,  DEXTRIN,  STARCH, 
PENTOSANS  AND  CELLULOSE. 

The  following  scheme  *  provides  for  each  of  the  substances  or 
groups  mentioned  and  avoids  the  danger  (inherent  in  the  usual 
plan  of  making  a  number  of  independent  determinations)  of 
including  the  same  substance  as  a  constituent  of  more  than  one 
group. 

Free  the  sample  from  fat  by  washing  with  ether. 

Extract  with  boiling  alcohol. 

Solution  A. —  Evaporate  the  alcohol,  dilute  with  water  and  deter- 
mine the  reducing  power  of  portions  of  the  solution  before  and 
after  hydrolysis.  Calculate  the  reducing  sugar  and  sucrose. 

Residue  A.  —  Extract  with  cold  water. 

Solution  B.  —  Hydrolyze  a  portion  and  determine  the  resulting 
dextrose,  calculate  dextrin  and  soluble  starch.  In  another  portion, 
precipitate  soluble  starch  by  barium  hydroxide,!  filter  and  deter- 
mine dextrin  in  the  filtrate. 

Residue  B.  —  Boil  with  water  and  treat  with  malt  extract  or 
saliva,  filter  and  wash  thoroughly. 

Solution  C.  —  Hydrolyze,  determine  resulting  dextrose  and  cal- 
culate starch. 

Residue  C.  —  Boil  with  2  percent,  hydrochloric  or  sulphuric  acid 
until  the  maximum  reducing  power  of  the  solution  is  reached. J 

Solution  D.  —  Determine  reducing  power  in  the  same  manner 
as  for  dextrose.  Calculate  as  xylose,  the  reducing  power  of  which 
is  1.03  times  that  of  dextrose.  §  From  the  pentose  thus  found  cal- 

*  Based  on  the  following  papers  :  Stone  ;  Journ.  Amer.  Chem.  Soc.,  1897,  19,  183. 
Sherman  ;  Ibid.,  1897,  19,  291.  Browne  and  Beistle ;  Ibid.,  1901,  23,  229. 

f  Asboth  :  Chem.  Ztg.,  1889,  13,  591. 

J  For  sulphuric  acid  this  was  found  to  be  4  to  6  hours.  Stone  prefers  hydrochloric 
acid  and  states  that  the  reaction  is  nearly  complete  in  I  hour. 

\  Stone  :  Amer.  Chem.  Journ.,  1891,  13,  82.  Since  the  reducing  power  of  arabin- 
ose  does  not  differ  greatly  from  that  of  xylose,  this  calculation  would  still  be  nearly 
correct  in  case  both  pentoses  were  present. 


loS  ORGANIC   ANALYSIS. 

culate  the  percentage  of  pentosan.  In  the  case  of  wheat  it  has 
been  found  *  that  the  material  ("  hemicellulose  ")  dissolved  and 
hydrolyzed  at  this  point  is  entirely  pentosan.  The  same  is  prob- 
ably true  of  the  other  cereals.  The  pentosan  thus  dissolved  and 
hydrolyzed  does  not  include  necessarily  the  entire  furfurol-yielding 
substance  of  the  cereal. 

Residue  D. —  Boil  for  30  minutes  with  I  per  cent,  sodium 
hydroxide,  filter  and  wash,  press  out  most  of  the  water  and  expose 
the  moist  fiber  to  chlorine  gas  for  one  hour.  Wash  with  water, 
boil  with  a  solution  containing  2  per  cent,  sodium  sulphite  and  0.2 
per  cent,  sodium  hydroxide ;  filter,  wash  with  warm  water  until  the 
washings  are  neutral  and  colorless,  then  wash  with  strong  alcohol, 
dry  and  weigh.  Deduct  the  ash  which  the  residue  contains  and 
calculate  the  organic  matter  as  cellulose,  f 

DETERMINATION  OF  MALTOSE,  DEXTRIN  AND  STARCH 
IN  MALTED  CEREAL. 

The  absence  of  any  considerable  amount  of  dextrose  or  invert- 
sugar  must  be  shown  by  stirring  some  of  the  sample  with  about 
ten  times  its  weight  of  water  and  testing  the  filtered  extract  by 
means  of  phenylhydrazine  or  Barfoed's  solution  as  described  in 
Chapter  V:  If  no  monosaccharide  is  present  the  percentages  of 
maltose,  dextrin  and  starch  can  be  estimated  as  follows: 

Mix  5  grams  of  sample  with  125  c.c.  of  cold  water \  in  a  250  c.c. 
flask ;  allow  to  stand  at  room  temperature  for  one  hour*  shaking 
frequently  ;  fill  to  the  mark,  shake  and  filter  through  dry  paper. 
Determine  reducing  power  of  one  or  more  25  c.c.  portions  of 
this  filtrate  by  either  Defren's  or  Allihn's  method  and  calculate 
the  amount  of  maltose. §  Measure  50  c.c.  of  the  same  filtrate  into 
a  100  c.c.  flask,  add  5  c.c.  of  hydrochloric  acid  of  1.125  SP-  gr-  and 
hydrolyze  as  in  the  determination  of  starch.  Determine  the  re- 
sulting dextrose,  deduct  the  amount  due  to  maltose  and  estimate 

*  Journ.  Amer.  Chem.  Soc.,  1897,  19,  294. 

f  This  is  the  method  of  Cross  and  Bevan.  For  a  comparison  of  this  with  other 
methods  see  Journ.  Amer.  Chem.  Soc.,  1897,  19,  304. 

J  If  the  sample  contains  an  active  enzyme  some  of  the  carbohydrate  may  be  changed 
during  this  treatment  with  water.  To  prevent  this  a  very  dilute  alkali  solution,  con- 
taining 0.02  per  cent,  potassium  hydroxide  or  an  equivalent  amount  of  sodium  or  am- 
monium hydroxide,  may  be  used.  Ling  and  Rendle  :  Journ.  Inst.  Brewing,  1904,  10, 
238  ;  Abs.  Jottrn.  Chem.  Soc.y  1904,  86,  ii.,  507. 

|  If  Allihn's  method  is  used,  assume  the  reducing  power  of  maltose  to  be  0.61 
that  of  dextrose. 


CARBOHYDRATES— SPECIAL    METHODS.        109 

the  remainder  as  due  to  dextrin.  Soluble  starch  if  present  would 
be  counted  as  dextrin  in  this  analysis. 

Treat  another  portion  of  the  original  sample  as  described  under 
the  determination  of  starch,  but  without  extracting  the  soluble  car- 
bohydrates. From  the  dextrose  found,  subtract  that  yielded  by 
maltose  and  dextrin  and  estimate  the  remainder  as  derived  from 
starch. 

The  results  require  a  slight  correction  on  account  of  the  pres- 
ence of  the  insoluble  residue  when  the  solution  is  diluted  to  vol- 
ume in  the  graduated  flask,  Although  some  details  of  the  method 
are  open  to  criticism,  it  gives  results  sufficiently  exact  for  the  pur- 
pose for  which  it  is  mainly  used,  which  is  to  show  whether  the 
starch  of  the  cereal  has  been  largely  changed  to  soluble  products. 

The  same  plan  may  be  used  in  the  examination  of  cereal  foods 
prepared  by  parching  or  in  other  ways,  provided  only  one  reducing 
sugar  is  present  in  appreciable  quantity.  The  amount  of  soluble 
carbohydrate  in  such  preparations  is  usually  too  small  for  satisfac- 
tory determination  by  means  of  the  polariscope. 

REFERENCES  TO  OTHER  SPECIAL  METHODS. 
SUBSTANCES  RICH  IN   SUCROSE  OR  INVERT-SUGAR. 

Detection  of  Sucrose. 

Wiley:  Agricultural  Analysis,  Volume  III.,  189.  Leach  :  Food 
Inspection  and  Analysis,  480. 

Determination  of  Sucrose,  Dextrose  and  Levulose. 
Wiechmann :  Journ.  Anal.  Appl.  Chem,,  1890,  4,  253;   1891,  5, 
401  ;  Ztschr.  anal.  Chem.,  1891,  30,  78.    Wiley:  /.  c.,  280-286,  307- 
308.      Vaubel :  Bestimmung  organischer  Verbindungen,  Band  II., 
447-450. 

Direct  Determination  of  Lwulose. 

Allen  :  Commercial  Organic  Analysis,  Vol.  I.  (3d  Ed.)  356. 
Wiley:  /.  c.,  267-274 ;  Journ.  Amer.  Chem.  Soc.,  1896,  18,  81. 

Determination  of  Sucrose  and  Commercial  Glucose. 
Chandler  and  Ricketts  :  Tucker's  Manual  of  Sugar  Analysis,  287. 
Weber  and  McPherson  :  Journ.  Amer.  Chem.  Soc.,  1895,  17,  312. 
Mathews  and  Parker:  Analyst,  1900,  25,  89. 
Leach:  /.  c.,  504-513;  Journ.  Amer.  Chem.  Soc.,  1903,  25,  982. 
Gudeman:  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  73,  p.  65. 


no  ORGANIC   ANALYSIS. 

Determination  of  Sucrose  and  Raffinose. 
Lippmann:  Chemie  der  Zuckerarten,  Band  II.,  1654-1661. 
Davoll :  Journ.  Amer.  Ghent.  Soc.,  1903,  25,  1019. 
Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

Determination  of  Moisture. 
Wiley:  Agricultural  Analysis,  Volume  III.,  13-36. 

Interpretation  of  Results. 

Molasses  and  Syrup.  —  Bui.  13,  Part  6,  Div.  Chem.,  U.  S.  Dept. 
Agriculture. 

Bodman,  Leonard  and  Smith:    Analyst,  1899,  24,  253. 

Jones :  Ibid.,  1900,  25,  87. 

Miller  and  Potts:  Journ.  Soc.  Chem.  Ind.,  1899,  18,  1091. 

Leonard:  Analyst,  1900,  25,  85. 

Maple  Products.  —  Leach,  /.  c.,  460,  767. 

Hortvet:  Journ.  Amer.  Chem.  Soc.,  1904,  26,  1523. 

Honey.  —  Bui.   13,  Part  6,  /.  c.     Leach  :  /.  c.,  5  1 1-514. 

Bujard  and  Baier  :   Hilf  buch  fur  Nahrungsmittel  Chemiker,  208. 

Beckmann  :  Ztschr.  anal.  Ckem.,  1896,  35,  263. 

Hoitsema:  Ibid.,  1899,38,  439. 

Circular  No.  13,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 

Confectionery.  —  Leach :  /.  c.,  5 1 8-5  2 1 ,  630. 

Bui.  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

ARTIFICIAL  MIXTURES  CONTAINING  LACTOSE. 

Qualitative  Examination. 

Bartley  and  Mayer:  Merck's  Report  1903,  12,  100.    Leach,  /.  c., 
507.     See  also  methods  given  in  Chapter  V. 

Determination  of  Sucrose  and  Lactose. 
In  milk  chocolate.  —  Laxa:  Ztschr.  Unters.Nahr.-Genussm.,  1904, 

7,471. 

In  condensed  milk.  —  Wiley :  /.  c.,  296-298. 
Hyde:J0urn.  Amer.  Chem.  Soc.,  1899,  21,  439. 
Riiber  and  Riiber:  Ztschr.  anal.  Chem.,-\gQ\,  40,  97. 
Harrison  :  Analyst,  1904,  29,  248. 

Determination  of  Sucrose,  Lactose  and  Invert- sugar. 
Bigelow  and  McElroy  :  Joutn.  Amer.  Chem.  Soc.,  1893,  15,  668. 


CARBOHYDRATES— SPECIAL    METHODS.         in 

Determination  of  Lactose  and  Maltose. 

Boyden  :  Journ.  Amer.  Chem.  Sec.,  1902,  24,  993.  See  also  mucic 
acid  method  for  lactose,  Chapter  V. 

ANIMAL  TISSUES  AND  FLUIDS  OTHER  THAN  MILK. 

Determination  of  Glycogen. 

Pfluger:  Arch.  ges.  Physiol.,  1899,  75,  120;  76,  531  ;   1904,  103, 
169;  Abs.Jwim.  Chem.  Soc.,  1904,  86,  ii,  595. 
Salkowski:  BiocJiem.  Centrbl.,  1903,  I,  337. 
Lebbin:  Pharm.  Ztg.,  1898,  43,  519;  Abs.  Chem.  Centrbl.,  1888, 

II.,  SIS- 

Huppert:  Ztschr.  physiol.  Chem.,  18,  137,  144. 
Hay  wood:  Journ.  Amer.  Chem.  Soc.t  1900,  22,85. 
U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  65,  p.   13. 

Detection  and  Determination  of  Dextrose. 

Hoppe-Seyler:  Physiologisch-  and  Pathologisch-Chemische  An- 
alyse (Revised  by  Thierfelder,  1903),  88-96. 

Vaubel:  Bestimmung  organischer  Verbindungen,  I.,  183,  501- 
503;  II.,  307,507-509. 

Allen:  /.  c.y  Volume  I.,  344-351.  Chemistry  of  Urine,  London, 
1895. 

Sutton  :  Volumetric  Analysis  (9th  ed.),  321,  409. 

Harley :  Journ.  Physiol. ,  1891,  12,  391. 

Raimann:  Ztschr.  anal.  Chem,  1901,  40,  390. 


CHAPTER  VIII. 

Acids. 

Free  organic  acids  when  dissolved  in  water  are  dissociated  to  a 
small  extent  and  can  usually  be  determined  by  direct  titration,  using 
as  standard  alkali  a  solution  of  a  strong  base  and  as  indicator  a 
substance  of  very  weak  acid  properties  such  as  phenolphthalein. 
In  cases  where  direct  titration  is  not  available,  the  determination 
of  acidity  can  be  made  by  an  indirect  method,  such  as  allowing  the 
substance  to  act  upon  a  carbonate  and  collecting  the  carbon  dioxide 
set  free,  or  upon  potassium  iodate  in  the  presence  of  an  excess  of 
iodide  when  each  carboxyl  hydrogen  entering  into  reaction  results 
in  the  liberation  of  one  atom  of  iodine.  The  iodine  thus  liberated 
is  determined  by  titration  with  standard  thiosulphate.  These  and 
other  methods  for  the  determination  of  the  carboxyl  group  are 
fully  discussed  by  Meyer  and  Tingle.*  Alkaline  salts  of  organic 
acids  are  usually  converted  into  carbonates  by  ignition,  so  that  the 
determination  of  carbonate  in  the  ash  of  a  neutral  or  neutralized 
mixture  often  indicates  the  amount  of  organic  acid  present  as  salt 
before  ignition. 

ACETIC  ACID  AND  ACETATES. 

Pure  acetic  acid  is  a  colorless  liquid  of  1.056  specific  gravity, 
miscible  in  all  proportions  with  water,  alcohol,  and  ether.  When 
nearly  anhydrous  it  solidifies  at  about  16°  —  hence  the  term  "  gla- 
cial "  as  applied  to  very  strong  acetic  acid.  The  pure  acid  boils 
without  decomposition  at  119°.  From  dilute  aqueous  solutions  it 
distils  readily  with  steam  at  the  temperature  of  boiling  water.  The 
principal  source  of  commercial  acetic  acid  is  the  crude  acetate  of 
lime  made  by  neutralizing  the  acid  obtained  in  the  dry  distillation 
of  wood.  For  the  determination  of  the  volatile  acid  obtainable 
from  commercial  acetate  the  following  method  can  be  used. 

DETERMINATION  OF  ACETIC  ACID  IN  CALCIUM  ACETATE. 
Arrange  a  round-bottomed  flask  of  300  c.c.  capacity  in  such  a 
way  that  it  can  be  inclined  at  an  angle  of  about  60°  from  the  per- 

*  Determination  of  Radicles  in  Carbon  Compounds. 

112 


ACIDS.  113 

pendicular  and  connected  with  a  nearly  vertical  condenser,  while 
another  tube  passing  through  the  stopper  of  the  flask  provides  for 
the  continuous  introduction  of  water,  drop  by  drop,  during  the  dis- 
tillation. The  flow  of  water  can  be  controlled  by  means  of  a  screw 
pinchcock  or  a  small  dropping  funnel. 

Weigh  2  grams  of  the  ground  sample,*  transfer  to  the  flask  and 
add  15  c.c.  of  50  per  cent,  phosphoric  acid  and  25  c.c.  of  water, 
being  sure  that  the  water  washes  down  any  of  the  sample  or  acid 
which  may  have  adhered  to  the  neck  of  the  flask.  Connect  the 
flask  with  the  condenser  and  distil,  collecting  the  distillate  in  a  re- 
ceiver containing  50  c.c.  of  water  to  prevent  loss  due  to  evaporation 
of  acetic  acid.  During  the  distillation  keep  the  volume  of  liquid 
nearly  constant  at  40  c.c.  by  admitting  water  free  from  carbon 
dioxide,  in  such  a  way  that  the  drops  fall  upon  the  inner  surface 
of  the  neck  of  the  flask  and  not  directly  into  the  boiling  liquid. 
Continue  the  distillation  until  the  distillate  is  no  longer  acid. 
This  usually  requires  about  one  and  one-half  hours.  Titrate 
the  distillate  with  freshly  standardized  sodium  hydroxide  solution 
using  phenolphthalein  as  indicator.  Calculate  the  total  acidity  as 
percentage  of  acetic  acid  in  the  sample. 

Notes.  —  It  has  been  found  that  small  amounts  of  phosphoric 
acid  are  frequently  carried  over  mechanically  if  the  acetic  acid  is 
removed  by  a  current  of  steam  or  if  the  distillation  is  conducted  in 
an  upright  flask,  especially  when  drops  of  water  are  allowed  to  fall 
directly  into  the  boiling  acid  mixture.  The  directions  for  arrange- 
ment of  apparatus  are  intended  to  avoid  this  source  of  error. 

The  phosphoric  acid  used  must  not  contain  nitric  or  any  other 
volatile  acid.  The  large  excess  recommended  dissolves  the  cal- 
cium phosphate  formed  and  thus  prevents  bumping.  Oxalic  may 
be  substituted  for  phosphoric  acid,  and  the  calcium  oxalate  re- 
moved by  filtration  before  distilling.  This  method,  however,  is 
longer  and  no  more  accurate  than  the  phosphoric  acid  method 
as  described.  Sulphuric  acid  cannot  be  used,  as  it  would  be  par- 
tially reduced  to  sulphurous  acid  by  the  tarry  matter  present  in 
the  crude  acetate. 

The  distillate  obtained  as  described  should  contain  only  a  minute 
amount  of  carbonic  acid.  It  is  frequently  recommended  that  the 
distillate  be  caught  in  standard  alkali  nearly  sufficient  to  neutralize 

*In  sampling,  grinding,  and  weighing  portions  for  analysis,  special  care  must  be 
taken  to  avoid  changes  in  moisture  content. 


ii4  ORGANIC  ANALYSIS. 

all  the  volatile  acid  expected.  In  this  case  the  distillate  would 
contain  all  carbonic  acid  liberated  in  the  distillation.  The  distil- 
late containing  some  free  acetic  acid  should  therefore  be  boiled 
under  a  reflux  condenser  to  expel  carbonic  acid  before  making  the 
final  titration.  Errors  may  also  be  caused  by  variation  in  the 
amount  of  carbonate  in  the  alkali  used  for  titration.  Hence  this 
must  be  freshly  standardized,  using  phenolphthalein  as  indicator, 
under  the  same  conditions  of  temperature  and  removal  of  carbon 
dioxide  as  exist  in  the  titration  of  the  distillate. 

The  volatile  organic  acid,  though  calculated  as  acetic,  always 
contains  some  formic,  propionic,  and  butyric  acids.  In  addition  to 
these  Scheuer  found  small  amounts  of  valerianic,  caproic,  heptylic, 
caprylic,  and  nonylic  acids.  In  view  of  the  danger  of  phosphoric 
acid  being  carried  over,  it  is  advisable,  after  titrating  the  distillate, 
to  add  nitric  acid,  evaporate  to  25  c.c.  and  test  for  phosphoric  acid 
by  adding  ammonium  nitrate  and  molybdate  solution. 

The  distillation  method  has  now  almost  entirely  replaced  the 
indirect  methods  formerly  used.  There  is,  however,  no  general 
agreement  as  to  the  details  of  manipulation.  A  full  description  of 
the  method  as  used  in  the  laboratory  of  the  General  Chemical 
Company  is  given  by  Grosvenor. 

References. 

Allen  :  Commercial  Organic  Analysis,  Volume  I. 
.  Sutton  :  Volumetric  Analysis. 

Scheuer  :  Analyse  von  Graukalk.  Dissertation,  Munich,  1902. 

Stillwell :  Acetic  Acid  in  Acetate  of  Lime.  Journ.  Soc.  Chem. 
Ind.9  1904,  23,  305. 

Grosvenor:  Analysis  of  Commercial  Acetate  of  Lime.  Ibid., 
1904,23,  530. 

SEPARATION  OF  ACETIC  ACID  FROM  ITS  HOMOLOGUES. 
It  was  explained,  in  connection  with  the  determination  of  alco- 
hols, that  methyl  alcohol  is  oxidized  to  carbon  dioxide  and  water 
by  heating  with  sulphuric  acid  and  potassium  dichromate  while 
ethyl  alcohol  is  oxidized  only  to  acetic  acid.  In  the  same  way 
formic  acid  can  be  determined  by  oxidation  with  the  dichromate 
mixture,  acetic  acid,  if  present,  being  unaffected  by  the  treatment. 
The  methods  of  Macnair  *  and  Freyerf  are  based  on  this  principle. 

*  Chem.  News,  1887,  55,  229. 
\Chem.  Ztg.,  1895,  19,  1184. 


ACIDS.  115 

According  to  Leys,  *  formic  acid  is  also  quantitatively  oxidized 
by  mercuric  acetate.  The  reaction  takes  place  readily  on  heating, 
even  in  very  dilute  solution,  and  is  said  to  yield  accurate  results 
which  are  not  affected  by  the  presence  of  acetic  acid,  alcohol,  or 
aldehyde. 

Aside  from  the  determination  of  formic  acid  by  oxidation,  the 
analysis  of  a  mixture  of  closely  related  homologues  is  very  diffi- 
cult. Haberland  proposed  f  a  method  for  the  separation  of  formic, 
acetic,  propionic,  and  butyric  acids  in  crude  wood  vinegar.  This 
method  involved  the  precipitation  of  propionic  acid  as  basic  lead 
propionate  insoluble  in  water,  and  of  formic  acid  as  zinc  formate 
insoluble  in  alcohol,  after  which  the  acetic  and  butyric  acids  were 
separated  either  by  the  difference  in  the  solubilities  of  the  silver 
salts  or  by  fractional  distillation  of  the  amyl  esters.  According  to 
Schiitz,J  however,  these  separations  are  far  from  quantitative. 

Wechsler§  separated  these  acids  qualitatively  by  the  method  of 
partial  distillation,  in  which  the  mixture  of  acids  is  treated  with  an 
amount  of  alkali  insufficient  for  complete  neutralization,  and  then 
distilled.  Under  these  conditions  the  higher  homologues,  although 
less  volatile,  are  concentrated  in  the  distillate,  the  lower  and 
stronger  acids  remaining  in  combination  with  the  base.  Scheuer|] 
applied  the  same  method  quantitatively  with  good  results  but 
found  it  too  long  for  practical  purposes. 

VINEGAR. 

A  thorough  examination  of  vinegar  should  include  determina- 
tions of  specific  gravity,  total  solids  or  extract,  total  and  soluble 
ash,  alkalinity  and  phosphoric  acid  of  the  ash,  total  and  volatile 
acidity,  and  alcohol.  Free  mineral  acids  and  oxalic  acid  should  be 
determined  if  present  and  tests  should  be  made  for  preservatives, 
poisonous  metals,  foreign  pungent  materials,  and  foreign  colors. 

Methods  for  all  of  these  determinations  have  been  adopted  by 
the  Association  of  Official  Agricultural  Chemists.  The  following 
directions  for  the  more  important  determinations  are  in  accordance 
with  the  official  methods. 

*  Bull.  Soc.  Chim.,  1898  [3],  19,  472;  Ghent.  News,  1898,  78,  245.  See  also 
Sparre  :  Ztschr.  anal.  Chem.,  1900,  39,  105. 

•\Ztschr.  anal.  Chem.,  1899,  38,  217. 

\  Ztschr.  anal.  Chem.,  1900,  39,  17. 

\Monatsh.  Chem.,  1893,  14,  462. 

||  Ueber  die  Trennungund  Bestimmung  fliichtiger  Fettsauren.  Dissertation,  Munich, 
1902. 


n6  ORGANIC  ANALYSIS. 

SOLIDS  AND  ASH. 

Total  Solids  or  Extract. 

Evaporate  10  c.c.  nearly  to  dryness  in  a  weighed  platinum  dish 
of  50  mm.  diameter  on  a  steam  bath,  dry  for  two  and  one-half 
hours  in  a  boiling  water  oven,  cool  thoroughly  in  a  desiccator  and 
then  weigh  quickly. 

Total  Ash. 

Char  the  extract  thoroughly  at  a  low  red  heat,  leach  with  water, 
burn  the  insoluble  residue  to  whiteness,  add  the  water  solution, 
evaporate  and  heat  to  low  redness,  cool  in  a  desiccator  and  weigh. 

Solubility  and  Alkalinity  of  Ash* 

Evaporate  25  c.c.  to  dryness,  burn,  cool  and  weigh ;  extract  the 
ash  repeatedly  on  an  ash-free  filter ;  dry  and  ignite  the  filter  and 
residue,  and  weigh  as  insoluble  ash.  Titrate  the  filtrate  using 
methyl  orange  as  indicator,  and  calculate  the  number  of  c.c.  of 
tenth-normal  acid  which  would  be  required  to  neutralize  the  cor- 
responding filtrate  from  100  c.c.  of  the  vinegar. 

TOTAL  AND  VOLATILE  ACIDS. 

Total  Acidity. 

Dilute  10  c.c.  in  a  beaker  until  the  solution  appears  very  light- 
colored  against  a  white  background,  add  phenolphthalein  and  titrate 
with  standard  sodium  hydroxide.  If  only  the  total  acidity  is  de- 
termined the  result  is  expressed  as  acetic  acid. 

Volatile  Acids. 

Heat  15  c.c.  of  the  vinegar  to  boiling  in  a  flask,  adding  a  little 
tannin  to  check  foaming,  if  necessary ;  lower  the  flame  and  distil 
with  steam  until  the  distillate  no  longer  contains  acid.  Titrate  the 
distillate  with  standard  sodium  hydroxide  and  calculate  as  acetic 
acid. 

The  difference  between  the  total  acidity  and  that  due  to  volatile 
acids  gives  a  measure  of  the  fixed  acids  and,  in  the  case  of  cider 
vinegar,  is  calculated  as  malic  acid. 

*  Smith's  method  modified  by  Frear  :  Journ.  Amer.  Chem.  Soc.,  1898,  20,  5  ;  Bui. 
65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


ACIDS.  117 

FREE  MINERAL  ACIDS. 

Detection  by  Methyl  Violet. 

Dilute  5  c.c.  of  the  vinegar  with  5  to  10  c.c.  water  to  reduce 
the  acidity  to  about  2  per  cent,  of  acetic  acid,  add  four  or  five 
drops  of  an  aqueous  solution  of  methyl  violet  (one  part  of  "  methyl 
violet  2B"  —  No.  56  of  Bayer  Farbenfabrik,  Elberfeld — in  10,000 
parts  of  water).  Mineral  acids  change  the  blue  violet  color  to  a 
blue  green  or  green. 

Determination  —  Hehner's  Method. 

To  a  weighed  quantity  of  the  sample,  add  a  measured  amount 
(more  than  sufficient  to  neutralize  all  mineral  acid  present)  of 
tenth-normal  alkali ;  evaporate  to  dryness,  incinerate  and  titrate 
the  ash  with  tenth-normal  acid  using  methyl  orange  as  indicator. 
The  difference  between  the  volume  of  alkali  added  and  that  of  acid 
required,  represents  the  equivalent  of  the  free  mineral  acid  in  the 
sample. 

DETERMINATION  OF  SOURCE.* 

The  principal  vinegar  of  the  United  States  is  cider  vinegar,  al- 
though considerable  quantities  of  malt  vinegar  are  also  used.  The 
substitutes  are  made  mainly  of  low-grade  wine  products,  sugar 
house  or  glucose  wastes,  and  wood  vinegar  from  acetate  of  lime. 
Cheap  apple  jelly  is  sometimes  added  to  the  substitutes  to  give 
them  the  color,  flavor,  and  body  of  cider  vinegar. 

Cider  vinegar  contains  from  1.2  to  8  per  cent,  of  solids,  average 
about  2.5  per  cent.;  malt  vinegar  1.75  to  6,  average  about  3  per 
cent.;  spirit  vinegar  rarely  over  0.75  per  cent,  average  about  0.30 
per  cent. 

The  total  ash  in  spirit  and  wood  vinegars  rarely  exceeds  o.  I  per 
cent.  In  fruit  and  malt  vinegars  it  rarely  falls  below  0.2,  cider 
vinegar  averaging  about  0.35  per  cent. 

The  alkalinity  of  the  ash,  expressed  as  above,  is,  according  to 
Frear,  for  cider  vinegar  26  to  65,  average  39;  for  malt  vinegar  5.5  ; 
for  spirit  vinegar  i.i. 

The  flame  reaction  of  the  solids  is  said  to  be  always  that  of  pot- 
ash in  the  case  of  cider  vinegar,  while  spirit,  sugar,  and  glucose 
vinegars  and  any  which  have  been  artificially  colored  show  the 
sodium  flame  (Davenport-Frear). 

*Cf.  Frear:  Bui.  65,  loc.  dt. 


n8  ORGANIC  ANALYSIS. 

The  optical  activity  of  vinegar  often  indicates  the  source.  Pure 
cider  vinegar,  after  clarification  with  basic  lead  acetate,  is  Isevoro- 
tatory,  a  200  mm.  tube  giving  usually  a  reading  of  —  0.5°  to  —  1.4° 
on  the  Ventzke  scale.  According  to  Browne,  levulose  is  the  only 
sugar  present  in  properly  fermented  cider  vinegar,  the  sucrose  and 
dextrose  having  both  disappeared  in  the  alcoholic  fermentation. 
Wine  vinegar  is  also  slightly  Isevorotatory.  Vinegar  from  sugar 
house  wastes  is  dextrorotatory  before,  and  Isevorotatory  after,  hy- 
drolysis. Glucose  vinegar  shows  dextrorotation  both  before  and 
after  hydrolysis.  Artificial  vinegars  can,  of  course,  be  made  laevoro- 
tatory  by  the  addition  of  apple  pomace. 

OFFICIAL  STANDARDS. 

The  definitions  and  standards  established  by  the  Secretary  of 
Agriculture  under  authority  of  Congress  are  in  part  as  follows :  * 

Vinegar  y  cider  vinegar,  or  apple  vinegar  is  the  product  made  by 
the  alcoholic  and  subsequent  acetous  fermentations  of  the  juice  of 
apples,  is  laevorotatory,  and  contains  not  less  than  4  grams  of  acetic 
acid,  not  less  than  1.6  grams  of  apple  solids,  and  not  less  than  0.25 
gram  of  apple  ash  in  looc.c.  The  water-soluble  ash  from  100  c.c. 
of  the  vinegar  neutralizes  not  less  than  30  c.c.  of  tenth-normal  acid 
and  contains  not  less  than  o.oio  gram  of  phosphoric  anhydride. 

Wine  or  grape  vinegar,  malt  vinegar,  sugar  vinegar,  glucose 
vinegar,  and  spirit  or  grain  vinegar  are  also  defined  with  special 
reference  to  the  materials  from  which  they  are  prepared  and  must 
all  contain  at  least  the  amount  of  acetic  acid  prescribed  under 
cider  vinegar. 

REFERENCES. 

Allen  :  Commercial  Organic  Analysis,  Volume  I. 

Leach  :  Food  Inspection  and  Analysis. 

Lunge :  Chemisch-technische  Untersuchungsmethoden,  Band  III. 

Browne  :  The  Chemical  Analysis  of  the  Apple  and  Some  of  Its 
Products,  Bui.  58,  Pennsylvania  Department  of  Agriculture; 
Journ.  Amer.  Chem.  Soc.t  1901,  23,  869. 

The  Effects  of  Fermentation  upon  the  Composition  of  Cider  and 
Vinegar,  Journ.  Amer.  Chem.  Soc.,  1903,  25,  16. 

Frear:  Vinegar,  Bui.  65,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

Leach  and  Lythgoe :  Cider  Vinegar  and  Suggested  Standards 
of  Purity,  Journ.  Amer.  Chem.  Soc.,  1904,  26,  375- 

*  U.  S.  Dept.  Agriculture,  Office  of  the  Secretary,  Circular  No.  13. 


ACIDS.  119 

Van  Slyke :  A  Study  of  the  Chemistry  of  Home-made  Cider 
Vinegar,  Bui.  258,  New  York  Agricultural  Experiment  Station, 

1904. 

FATTY   ACIDS. 

The  fatty  acids  are  all  monobasic  *  and  those  of  the  common 
fats  all  contain  an  even  number  of  carbon  atoms  in  the  molecule. 
Fatty  acids  containing  odd  numbers  of  carbon  atoms  are  not 
widely  distributed  in  nature,  being  usually  characteristic  of  some 
particular  fat  or  small  group  of  fats.  The  following  statement  of 
analytical  properties  is  based  mainly  upon  the  data  given  by 
Lewkowitsch.f 

ACIDS  OF  THE  SERIES  CnH2nO2. 

Butyric  acid,  ^HgO,  occurs  J  in  butter  and  in  very  small  quan- 
tity in  a  few  other  fats.  It  is  a  mobile  liquid,  mixing  in  all  pro- 
portions with  water,  alcohol,  and  ether.  It  boils  without  decompo- 
sition at  162°  and  is  readily  volatile  with  steam.  Specific  gravity 
at  20°,  0.959. 

Caproic  acid,  C6H12O2,  occurs  in  butter  and  cocoanut  fat.  It  is  a 
rather  oily  liquid  of  unpleasant  odor,  not  miscible  with  water  but 
somewhat  soluble  in  it  and  volatile  with  steam.  Boiling  point 
about  200°.  Specific  gravity  at  20°,  0.924. 

Caprylic  acid,  C8H16O2,  occurs  in  cocoanut  oil,  butter,  and  human 
fat.  In  the  cold  it  crystallizes  in  leaflets  which  melt  at  16.5°.  It 
boils  at  237°  and  is  volatile  with  steam.  One  part  dissolves  in 
400  parts  of  boiling  water  from  which  it  separates  almost  com- 
pletely on  cooling.  It  is  readily  soluble  in  alcohol  and  ether. 
Specific  gravity  at  20°,  0.910. 

Capnc  acid,  C10H20O2,  has  been  found  chiefly  in  cocoanut  oil,  but- 
ter, and  the  fat  of  the  spice  bush,  Lindera  benzoin.  It  melts  at 
31.3°,  boils  at  270°,  and  distils  with  steam.  Soluble  in  about 
1,000  parts  of  boiling  water;  almost  insoluble  in  cold  water;  solu- 
ble in  alcohol  and  ether. 

Laurie  acid,  C12H24O2,  occurs  abundantly  in  the  fat  of  the  seeds 
of  Lindera  benzoin,  and  in  smaller  proportions  in  butter,  cocoanut 
fat,  palm  oil,  laurel  oil,  and  the  nut  oil  of  the  California  bay  tree. 
Laurie  acid  melts  at  43.6°  and  cannot  be  distilled  at  atmospheric 
pressure  without  decomposition.  It  is  practically  insoluble  in  cold 

*  For  a  possible  exception  see  Gietel  and  Want :  Journ.  prakt.  Chem. ,  1900,  61,  151. 
fOils,  Fats,  and  Waxes  (Third  Edition). 

\  These  statements  refer  to  the  occurrence  of  the  acids  as  esiers  rather  than  in  the 
free  state- 


120  ORGANIC  ANALYSIS. 

water,  slightly  soluble  in  boiling  water  and  appreciably  volatile 
with  steam.  Specific  gravity  at  20°,  0.883. 

Myristic  acid,  CuH28O2,is  found  in  nutmeg  butter,  dika  fat,  butter, 
cocoanut  oil,  lard,  and  many  other  fats,  as  well  as  in  spermaceti  and 
wool  wax.  It  crystallizes  in  leaflets  which  melt  at  53.8°.  Myris- 
tic acid  is  insoluble  in  water  and  only  very  slightly  volatile  with 
steam.  It  is  not  readily  soluble  in  cold  alcohol. 

Palmitic  acid,  CI6H32O2,  occurs  in  nearly  all  solid  fats  and  non- 
drying  oils  as  well  as  in  several  waxes  including  spermaceti  and 
beeswax.  It  melts  at  62.6°  and  solidifies  on  cooling  to  a  scaly 
crystalline  mass.  It  is  insoluble  in  water  and  not  readily  soluble 
in  cold  95  per  cent,  alcohol,  but  dissolves  in  about  10  parts  of  cold 
absolute  alcohol. 

Steaiic  acid,  C18H36O2,  is  found  in  most  fats  and  occurs  most 
abundantly  in  those  having  high  melting  points.  It  crystallizes  in 
leaflets  which  melt  at  69.3°.  It  is  less  soluble  in  alcohol  than 
palmitic  acid,  requiring  about  40  parts  of  absolute  alcohol  in 
the  cold.  It  dissolves  readily  in  ether,  benzol,  carbon  bisulphide, 
or  hot  alcohol. 

Arachidic  acid,  C20H40O2,  occurs  in  arachis  (peanut)  oil  and  has 
been  obtained  in  very  small  quantities  from  several  other  fats. 
Arachidic  acid  is  distinguished  from  all  its  lower  homologues  by 
its  insolubility  in  cold  alcohol.  It  dissolves  freely  in  hot  alcohol 
but  on  cooling  separates  almost  completely  as  needles  or  crystal- 
line scales,  which  melt  at  77°  to  78°. 

Behenic  acid,  C22HUO2,  lignoceric  acid,  C24H48O2,  and  an  isomer  of 
the  latter,  carnaubic  acid,  occur  respectively  in  oil  of  ben,  arachis 
oil,  and  carnaiiba  wax. 

Cerotic  acid,  C26H52O2,  and  melissic  acid,  C30H60O2,  are  found  in 
the  free  state  in  beeswax. 

The  change  in  properties  of  these  acids,  as  the  molecular  weight 
increases,  is  noticeably  regular.  The  melting-  and  boiling-points 
rise  while  the  specific  gravities  and  solubilities  decrease.  In  gen- 
eral the  properties  of  the  glycerides  and  other  esters  vary  in  the 
same  way  as  those  of  the  free  acids. 

With  the  exception  of  caproic,  all  of  the  acids  of  this  series,  oc- 
curring in  natural  fats,  are  believed  to  be  of  the  "  normal "  (straight 
chain)  structure. 

ACIDS  OF  THE  SERIES  CnH2n_2O2. 
These  acids  are  unsaturated.    Each  molecule  contains  one  ethy- 


ACIDS.  121 

lene  linkage  or  double  bond  and  is,  therefore,  capable  of  taking 
up  by  direct  addition  two  atoms  of  halogen  or  one  molecule  of 
hydrobromic  acid  to  form  a  saturated  compound.  Careful  oxida- 
tion in  the  presence  of  moisture  results  in  the  formation  of  the  cor- 
responding dihydroxy-compounds.  The  unsaturated  acids  have, 
as  a  rule,  lower  melting  points  than  the  saturated  acids  containing 
the  same  number  of  carbon  atoms,  and  are  therefore  found  more 
largely  in  oils  and  soft  fats.  Only  the  more  important  members 
of  the  series  will  be  considered  here. 

Hypogceic  add,  C16H30O2,  obtained  from  arachis  (peanut)  oil,  melts 
at  33°,  but  is  converted  by  the  action  of  nitrous  acid  into  the 
isomeric  gaidic  acid  which  melts  at  39°.  Phycetoleic  acid,  isomeric 
with  hypogseic,  is  obtained  from  sperm  oil  and  seal  oil.  It  melts 
at  30°  and  is  not  changed  by  nitrous  acid. 

Oleic  acid,  C18H34O2,  occurs  in  nearly  all  fats  and  fatty  oils.  It  is 
an  oily  liquid  which  solidifies  at  4°  and  melts  at  14°.  Nitrous 
acid  converts  oleic  acid  into  the  isomeric  elaidic  acid  which  is  a 
crystalline  solid  melting  at  44.5°  (Lewkowitsch). 

Erucic  acid,  CMH42O2,  found  in  rape  seed  and  mustard  seed  oils, 
melts  at  33°  to  34°.  By  the  action  of  nitrous  or  sulphurous  acid 
it  can  be  converted  into  brassidic  acid,  melting  at  65°.  This 
change,  however,  takes  place  less  readily  than  the  corresponding 
conversion  of  oleic  acid  into  elaidic  acid. 

The  gradual  change  in  properties  with  increasing  molecular 
weight,  noticed  in  the  saturated  acids,  is  not  apparent  in  this  series, 
doubtless  because  the  known  acids  of  the  series  differ  as  regards 
the  position  of  the  double  bond  and  are  therefore  not  strictly  simi- 
lar in  constitution.  The  property  of  changing  to  solid  isomers 
under  the  influence  of  nitrous  acid  is  characteristic  of  the  acids  of 
this  series  and  furnishes  a  means  of  testing  for  them  in  fatty  oils. 
Any  of  the  latter  which  contain  large  amounts  of  these  acids  be- 
come solid  or  semi-solid  on  treatment  with  nitrous  acid.  This  is 
known  as  the  elaidin  test  and  is  sometimes  useful  in  the  preliminary 
examination  of  oils,  but  has  been  shown  to  be  less  reliable  than 
was  formerly  supposed. 

An  important  characteristic  of  the  unsaturated  acids  is  the  solu- 
bility of  the  lead  soaps  in  ether.  Muter's  method  for  the  separa- 
tion of  saturated  and  unsaturated  fatty  acids,  is  to  precipitate  the 
mixture  of  acids  as  lead  soaps  and  wash  with  ether.  While  this 
method  is  not  strictly  quantitative,  it  is  probably  the  best  which 
has  been  devised  for  the  purpose. 


122  ORGANIC  ANALYSIS. 

ACIDS  OF  THE  SERIES  CnH2n_4O2. 

The  acids  of  this  series  unite  with  four  atoms  of  bromine  or 
iodine  and  absorb  oxygen  on  exposure  to  the  air. 

Linoleic  acid,  C18Hg2O2,  is  the  only  important  member  of  the  series. 
It  is  widely  distributed  occurring  most  abundantly  in  the  "  dry- 
ing oils,"  so  called  because  they  are  oxidized  to  solids  on  exposure 
to  air.  While  especially  characteristic  of  the  drying  and  semi- 
drying  oils,  linoleic  acid  is  also  found  in  nondrying  oils  and  in  small 
quantity  in  some  of  the  solid  fats.  It  absorbs  oxygen  from  the 
air  so  readily  as  to  interfere  seriously  with  ordinary  methods  of 
purification  and  examination.  Its  physical  properties  are  therefore 
not  well  known  but  it  has  been  shown  to  have  a  higher  specific 
gravity  than  oleic  acid  and  a  much  lower  melting  point  since  it 
does  not  solidify  at  —  18°.  Linoleic  acid  is  not  changed  by  nitrous 
acid.  Lead  linoleate  is  soluble  in  ether. 

ACIDS  OF  THE  SERIES  CnH2n_^O2. 

Linolenic  acid,  C18H30O2,  is  the  only  member  of  this  series  which 
has  been  obtained  in  the  free  state.  It  is  a  colorless  oil  which 
absorbs  oxygen  very  rapidly  from  the  air,  at  the  same  time  becom- 
ing dark  brown.  The  purest  preparations  of  the  acid  which  have 
been  described  absorbed  242  to  245  per  cent,  of  iodine.  The  pure 
acid  should  absorb  six  atoms  or  274  per  cent,  of  iodine.  Linolenic 
acid  has  not  been  solidified.  Its  specific  gravity  is  greater  than 
that  of  linoleic  acid.  The  lead  soap  is  soluble  in  ether.  Linolenic 
acid  occurs  in  considerable  quantity  in  linseed  oil.  By  oxidizing 
linseed  oil  acids  with  alkaline  permanganate  Hazura  obtained  two 
acids  of  the  formula  C^H^OH)^,  linusic  and  isolinusic  acids, 
and  hence  inferred  the  presence  of  an  isolinolenic  acid  in  linseed  oil. 
Jecoric  acid,  isomeric  with  linolenic  acid,  is  reported  by  Fahrion  * 
as  existing  in  sardine  oil.  Since  the  oils  of  fish  and  other  marine 
animals  do  not  dry  like  the  vegetable  oils  having  the  same  iodine 
absorbing  power,  it  is  probable  that  the  highly  unsaturated  acids 
of  the  former  oils  are  in  general  isomeric,  rather  than  identical, 
with  linolenic  acid.  Some  of  the  fish  oils  are  thought  to  contain 
acids  of  the  series  CnH2n_tO2 

SATURATED  HYDROXY-ACIDS. 

When  any  of  the  unsaturated  acids  occurring  in  vegetable  oils 
is  carefully  oxidized  in  alkaline  solution  by  means  of  potassium 

*Chem.  Ztg.,  1893,  17,521. 


ACIDS.  123 

permanganate,  hydroxyl  is  added  until  the  molecule  becomes  sat- 
urated. Thus  oleic  acid  yields  dihydroxystearicacid,  C18H34(OH)2O2) 
linoleic  acid  yields  sativic  acid,  C18H32(OH)4O2,  and  linolenic  acid 
yields  linusic  acid,  C18H30(OH)6O2.  Similar  changes  in  the  unsat- 
urated  acids  may  take  place  when  their  glycerides  are  exposed  to 
the  air.  Small  amounts  of  saturated  hydroxy-acids  are,  therefore, 
likely  to  be  present  in  fatty  oils  which  have  been  kept  for  some 
time  in  partially  filled  bottles  or  otherwise  exposed  to  atmospheric 
oxidation.  Only  two  acids  of  this  series  have  as  yet  been  found  in 
nature.  Juillard*  found  about  I  per  cent,  of  dihydroxystearic  acid 
in  castor  oil  and  Brownef  the  same  quantity  in  butter  fat.  A 
dihydroxy-acid  of  the  formula,  C30H60O4,  is  said  to  occur  in  wool 
wax;};  and  is  called  lanocerinic  or  lanoceric  acid. 

HYDROXY-ACID  OF  THE  SERIES  C/iH2n_2O3. 

Ricinoleic  acid,  C18H34O3,  the  only  important  member  of  this 
series,  occurs  in  large  quantity  in  castor  oil.  It  is  a  very  viscous 
liquid  of  much  higher  specific  gravity  than  oleic  or  linoleic  acid. 
According  to  Juillard§  the  pure  acid  melts  at  4°-5°,  and  is  miscible 
with  alcohol  and  ether  in  all  proportions.  Ricinoleic  acid  is  dextro- 
rotatory. The  position  of  the  asymetric  carbon  atom,  as  well  as 
of  the  double  bond,  is  shown  by  the  following  formula]] 

C6H13  •  CH(OH)  •  CH2  •  CH  :  CH  (CH2)7  -  COOH. 

Ricinoleic  resembles  oleic  acid  in  its  chemical  reactions.  It  ab- 
sorbs two  atoms  of  bromine  or  iodine.  By  treatment  with  per- 
manganate in  alkaline  solution  two  hydroxyl  radicals  are  added. 
By  treatment  with  nitrous  acid,  ricinoleic  acid  is  converted  into  the 
solid  isomer,  ricinelaidic  acid.  Lead  ricinoleate  is  easily  soluble  in 
ether. 

SEPARATION  OF  FATTY  ACIDS. 

For  the  analytical  separation  of  saturated  from  unsaturated  fatty 
acids,  Muter's  method,  based  on  the  solubility  of  the  lead  soaps  of  the 
latter  in  ether,  is  generally  used.  In  order  to  determine  the  con- 
stituents of  a  mixture  of  homologous  acids  it  is  usually  necessary  to 

*  Bull.  Soc.  Chim.,  1895,  [3]  13,  238. 
f  Journ.  Anter.  Chem.  Soc.,  1899,  ax,  817. 

J  Darmstaedter  and  Lifschiitz  :  Ber.  deut.  chem.  Ges.,  1896,  29,  1476. 
g  Bull.  Soc.  Chim.,  1895,  [3],  13,  240. 

||Goldsobel:  Ber.  deut.  chem.  Ges.,  1894,27,  3121.  Kasansky  :  Journ.  prakt. 
Chem.,  1900,  62,  363. 


124  ORGANIC  ANALYSIS. 

fraction  the  mixture  repeatedly  either  by  distillation,  precipitation  f 
or  crystallization,  until  each  fraction  contains  only  two  acids.  The 
proportions  of  the  two  constituents  can  then  be  found  by  determin- 
ing the  mean  molecular  weight  of  the  mixture.  Such  methods  are 
outlined  by  Allen  (Commercial  Organic  Analysis,  3d  Ed.,  I.,  485- 
492  ;  II.,  240-246)  and  by  Browne,  in  a  paper  on  the  chemistry  of 
butter  fat,  Journ.  Amer.  Chem.  Soc.t  1899,  21,  807. 


CHAPTER  IX. 

Oils,  Fats,  and  Waxes  —  General  Methods. 

All  glycerides  of  fatty  acids  are  properly  known  as  fats.  As  a 
matter  of  convenience  those  fats  which  are  liquid  at  ordinary  tem- 
peratures are  commonly  called  fatty  oils.  Fats  and  fatty  oils,  there- 
fore, constitute  a  definite  group  of  compounds,  the  glyceryl  esters 
of  the  fatty  acids. 

Waxes  are  esters  of  fatty  acids  with  monatomic  alcohols  of  high 
molecular  weight.  Most  waxes  are  solid  at  ordinary  temperatures 
and  the  term  is  sometimes  applied  to  other  solids  of  similar  physical 
properties,  solid  hydrocarbons,  for  example,  being  frequently  called 
mineral  waxes. 

The  term  oil  has  no  strict  chemical  significance,  being  applied 
not  only  to  liquid  fats  but  also  to  substances,  such  as  mineral  and 
essential  oils,  which  are  similar  in  some  physical  properties  but  en- 
tirely different  in  constitution.  The  mineral  oils  are  conveniently 
treated  in  connection  with  the  fatty  oils  in  works  on  technical  analy- 
sis, because  on  account  of  their  similar  physical  properties  they  may 
for  certain  uses  be  mixed  with,  or  substituted  for,  the  fatty  oils  and 
are  sometimes  found  as  adulterants  of  the  latter.  The  same  is  true 
of  some  of  the  essential  oils,  notably  rosin  oil  and  turpentine. 

The  methods  given  in  this  chapter  are  described  and  discussed 
with  special  reference  to  the  analysis  of  solid  and  liquid  fats.  In 
most  cases,  however,  they  are  also  applicable  to  waxes,  non- fatty 
oils,  and  technical  mixtures. 

PROPERTIES  OF  FATS  AND  FATTY  OILS. 

Refined  fats  and  fatty  oils  are  usually  light  yellow  to  colorless. 
Vegetable  oils  are  sometimes  tinged  green  by  the  presence  of 
chlorophyll.  Crude  oils  are  often  reddish  or  even  dark  brown. 
The  characteristic  colors,  odors,  and  flavors  of  natural  fats  are  due 
to  small  quantities  of  substances  other  than  glycerides,  and  there- 
fore become  less  perceptible  the  more  thoroughly  the  oil  is  refined. 
It  is  believed  that  all  of  the  natural  glycerides  except  butyrin,  if 
obtained  absolutely  pure,  would  be  colorless,  tasteless,  and  odorless. 

125 


126  ORGANIC  ANALYSIS. 

The  natural  glycerides  are  all  lighter  than  water  and  insoluble  in 
it.  They  can  take  up  a  very  small  amount  of  water,  which  is  given 
off  as  steam  on  heating.  The  quantity  of  water  which  can  be  held 
by  a  fatty  oil  without  causing  turbidity  is,  however,  negligible,  so 
that  for  practical  purposes  the  fatty  oils  may  be  considered  as 
immiscible  with  water. 

They  dissolve  readily  in  ether,  carbon  bisulphide,  chloroform, 
carbon  tetrachloride,  and  benzol,  and  mix  with  each  other  in  all 
proportions.  With  the  exception  of  castor  oil  and  a  few  other  oils 
characterized  by  a  large  proportion  of  hydroxy-acids,  they  are 
sparingly  soluble  in  alcohol  or  acetic  acid,  but  dissolve  readily  in 
petroleum  ether  and  mix  in  all  proportions  with  mineral  oils.  Cas- 
tor oil  is  readily  soluble  in  alcohol  or  acetic  acid  and  not  readily 
miscible  with  petroleum  ether  or  mineral  oils. 

The  natural  fats  do  not  distil  without  decomposition.  When 
decomposed  by  heating  they  give  off  acrolein  which  is  readily 
recognized  by  its  characteristic  irritating  odor. 

A  simple  triglyceride  is  one  in  which  the  three  acid  radicals  are 
of  the  same  kind.  A  glyceryl  ester  containing  the  radicals  of  two 
or  three  different  fatty  acids  is  known  as  a  mixed  glyceride.  Both 
simple  and  mixed  glycerides  have  been  isolated  from  natural  fats 
and  certain  physical  differences  in  fats  which  contain  practically 
the  same  acids  are  now  attributed  to  the  presence  of  mixed 
glycerides.  For  discussions  of  simple  and  mixed  triglycerides, 
see  Lewkowitsch's  Oils,  Fats,  and  Waxes,  3d  Ed.,  Chapter  I.,  and 
recent  papers  by  Hansen,*  Holde,f  and  Kreis  and  Hafner.J 

ANALYTICAL   METHODS. 

The  object  of  an  ordinary  fat  or  oil  analysis  is  not  so  much  to 
separate  individual  constituents  as  to  determine  certain  chemical 
and  physical  properties  which  are  fairly  constant  for  each  variety 
when  pure  and  are  therefore  frequently  called  analytical  constants. 
All  solid  and  liquid  fats  being  essentially  mixtures  of  triglycerides, 
any  differences  in  chemical  and  physical  properties  (except  such 
physical  variations  as  are  due  to  mixed  glycerides)  must  be  attrib- 
uted mainly  to  the  presence  of  different  fatty  acids,  or  of  the 
same  acids  in  different  proportions. 

*  Ueber  das  Vorkommen  gemischter  Fettesaiire-Glyceride  in  theirischen  Fette. 
Dissertation,  Rostock,  1902;  Arch.  Hygiene,  1902,  42,  I. 

f  Ber.  deut.  chem.  Ges.,  1902,  35,  4306. 

\  Ber.  deut.  chem.  Ges.,  1903,  36,  1123,  2766  ;  Ztschr.  Unters.  Nahr.-Genussm., 
1904,  7,  641. 


OILS,   FATS,   AND    WAXES.  127 

The  principal  differences  to  be  expected  are:  (i)  In  the  mean 
molecular  weight  of  the  acids  present  or  the  relative  proportions 
of  acids  of  high  and  those  of  low  molecular  weight ;  (2)  in  the 
relative  number  of  "double  bonds"  depending  upon  the  propor- 
tions of  acids  of  the  stearic,  oleic,  linoleic,  and  linolenic  types;  (3) 
in  the  proportion  of  hydroxy-acids  present. 

Some  of  the  analytical  "  constants  "  express  direct  measures  of 
one  of  these  three  properties.  Others,  especially  the  physical  con- 
stants, are  influenced  by  variation  in  any  of  these  three  directions 
and  therefore  express  no  one  chemical  property  but  a  resultant 
of  all.  The  "constants"  most  used  may  be  grouped  on  this 
principle  as  follows : 

1 .  (a)  Measuring  the  mean  molecular  weight. — Saponification  or 
Koettstorfer  number,     (b]  Measuring  the  proportion  of  acids  of 
high  or  of  low   molecular   weight. —  Hehner  number,  Reichert- 
Meissl  number. 

2.  (a)  Measuring  the  proportion  of  unsaturated  acids  (number 
of  "  double  bonds  "). — Hubl  number  and  other  halogen  absorption 
numbers,     (b]  Depending  mainly  upon  the  proportion  of  unsatu- 
rated acids. —  Thermal  reactions  with  bromine  or  sulphuric  acid, 
Maumene  number. 

3.  Measuring   the   hydroxyl   radical    and  therefore  depending 
mainly  upon  the  presence  of  hydroxy-acids. —  Acetyl  number. 

4.  Influenced  by  all  of  the  above  properties. —  Specific  gravity, 
index   of  refraction,  melting    point,  "  titer  test,"  viscosity,  solu- 
bilities. 

THE  SAPONIFICATION  OR  KOETTSTORFER  NUMBER.* 
The  saponification  or  Koettstorfer  number  is  the  number  of 
milligrams  of  potassium  hydroxide  consumed  in  the  complete 
saponification  of  one  gram  of  the  fat  or  wax ;  or,  in  other  words, 
it  is  ten  times  the  percentage  of  potassium  hydroxide  required  to 
neutralize  the  total  fatty  acids  in  the  sample,  whether  free  or  in  the 
form  of  esters. 

Reagents. —  I.  Standard  solution  of  hydrochloric  acid  prefer- 
ably half-normal. 

2.  Alcoholic  potash  solution  containing  40  grams  of  potassium 
hydroxide  per  liter  of  purified  95  per  cent,  alcohol.  In  the  prep- 

*  Koettstorfer  :  Ztschr.  anal.  Chem.t  1879,  18,  199,  431.  Reprinted  in  Ephraim's 
Originalarbeiten  iiber  Analyse  der  Nahrungsmittel,  Leipzig,  1895. 


128  ORGANIC  ANALYSIS. 

aration  of  this  solution  the  best  available  potassium  hydroxide 
(purified  by  alcohol)  should  be  dissolved  in  alcohol  which  has 
been  purified  by  redistillation  over  caustic  alkali.  The  solution 
must  be  clear  when  used. 

3.  As  indicator  a  I  per  cent,  solution  of  phenolphthalein  in  puri- 
fied 95  per  cent,  alcohol. 

Determination.  —  Weigh  4  to  5  grams  of  the  fat  or  oil  in  a  250 
c.c.  Erlenmeyer  flask,  add  50  c.c.  of  the  alcoholic  potash  solution, 
connect  with  a  reflux  condenser  and  boil  for  thirty  minutes,  or 
until  the  oil  is  completely  saponified,  so  that  the  liquid  in  the  flask 
appears  homogeneous  and  clear.  At  the  same  time  measure  50 
c.c.  of  the  alcoholic  potash  solution  into  an  empty  flask  of  the  same 
size  and  shape,  connect  with  a  similar  reflux  condenser  and  boil  for 
the  same  length  of  time  as  in  the  case  of  the  solution  containing  the 
sample.  Cool  the  flasks  and  titrate  each  with  the  standard  hydro- 
chloric acid,  using  I  c.c.  of  the  phenolphthalein  solution  as  indi- 
cator. The  difference  between  the  titrations  gives  a  measure  of  the 
potassium  hydroxide  consumed  in  saponifying  the  sample. 

Notes.  —  When  ordinary  alcohol  is  used  for  the  potassium  hy- 
droxide solution,  the  latter  rapidly  turns  brownish  so  that  the  final 
titration  is  difficult.  Alcohol  which  has  been  treated  with  potas- 
sium hydroxide,  allowed  to  stand  for  one  to  two  weeks  and  then 
redistilled,  gives  a  much  more  permanent  solution.  According  to 
Gill  scarcely  any  darkening  of  the  solution  occurs  if  it  is  kept  under 
an  atmosphere  of  hydrogen.  Great  care  must  be  exercised  to 
measure  exactly  the  same  quantity  of  the  alkaline  solution  into 
each  flask,  and  to  treat  the  blank  solution  in  exactly  the  same  way  as 
that  containing  the  sample,  so  that  any  loss  of  alkalinity  due  to 
absorption  of  carbon  dioxide  from  the  air,  or  to  the  possible  action 
of  the  alkali  on  the  solvent,  may  be  the  same  in  each  case.  If  sul- 
phuric acid  were  used  in  place  of  hydrochloric  for  the  final  titration 
a  precipitate  of  potassium  sulphate  would  be  formed  in  the  alco- 
holic solution,  thus  impairing  the  delicacy  of  the  end  reaction. 

In  order  to  avoid  the  changes  which  may  occur  in  the  alkali 
solution  on  boiling,  Henriques  *  recommends  that  the  sapon- 
ification  be  conducted  in  the  cold.  From  3  to  4  grams  of  oil  are 
mixed  with  25  c.c.  of  petroleum  ether  and  25  c.c.  of  normal  alco- 
holic potash  and  allowed  to  stand  over  night  at  room  temperature, 
when  the  saponification  is  said  to  be  complete. 

*  Ztschr.  angew.    Chem.,  1895,  721  ;   1896,  221,  423. 


OILS,   FATS,   AND    WAXES.  129 

The  Saponification  Equivalent. 

The  results  obtained  as  described  are  sometimes  expressed  in 
terms  of  the  saponification  equivalent.  This  is  the  weight  of  fat 
which  reacts  with  the  molecular  weight  of  sodium  or  potassium 
hydroxide ;  or,  in  other  words,  the  number  of  grams  of  fat  which 
would  be  saponified  by  one  liter  of  normal  alkali.  Comparing 
this  with  the  definition  of  the  saponification  (or  Koettstorfer)' #&;;*- 
her  it  will  be  seen  that  these  two  values  express  the  same  property 
in  reciprocal  terms  and  that  the  product  of  the  two  values  is 
always  equal  to  the  number  of  milligrams  of  potassium  hydrox- 
ide in  a  liter  of  normal  solution,  viz.  56,158. 

Hence 

56,158 

Saponification  equivalent  =  -      — r^ — -. —       — r— 

saponification  number 

56,158 

Saponification  number       =  -     — r^ : —      — ; — ; — 

saponification  equivalent 

For  a  sample  consisting  entirely  of  triglycerides  the  saponification 
equivalent  would  be  exactly  one-third  of  the  mean  molecular 
weight  of  these  glycerides. 

Acid  and  Ester  Numbers. 

The  acid  number  of  a  fat  or  wax  is  the  number  of  milligrams  of 
potassium  hydroxide  required  to  neutralize  the  free  fatty  acids  in 
one  gram  of  substance.*  The  ester  number  is  the  difference  be- 
tween the  saponification  number  and  the  acid  number  and  there- 
fore shows  the  amount  of  alkali  consumed  in  the  saponification  of 
esters. 

To  determine  the  acid  number,  shake  4  to  5  grams  of  the  sample 
with  50  c.c.  of  carefully  neutralized  alcohol  and  titrate  with  tenth- 
normal  or  half-normal  alkali,  using  phenolphthalein  as  indicator. 
As  the  oil  itself  mixes  but  slightly  with  alcohol,  it  is  necessary 
toward  the  end  of  the  titration  to  shake  thoroughly  after  each 
addition  of  alkali  to  secure  complete  extraction  of  the  fatty  acid 
from  the  oily  layer. 

*The  acidity  of  a  sample  of  oil  or  fat  is  not  always  expressed  as  the  "  acid  num- 
ber." Frequently  it  is  recorded  in  terms  of  the  equivalent  percentage  of  free  oleic 
acid,  "acidity  as  oleic,"  and  sometimes  as  "degrees  of  acidity."  The  latter  term 
indicates  the  number  of  c.c.  of  normal  caustic  alkali  required  to  neutralize  the  free 
acids  in  loo  grams  of  the  fat. 


130  ORGANIC  ANALYSIS. 

THE  HEHNER  NUMBER. 

The  Hehner  number  is  the  percentage  of  insoluble  fatty  acids 
obtainable  from  a  fat. 

As  the  determination  is  ordinarily  made,  the  unsaponifiable 
matter  present  in  the  fat  is  weighed  with  the  insoluble  acids.  The 
great  majority  of  fats  and  fatty  oils  have  Hehner  numbers  between 
94.5  and  96.  Butter  fat  has  a  lower  Hehner  number  and  the  de- 
termination of  this  "  constant "  is  of  value  chiefly  in  testing  the 
purity  of  butter.  The  detailed  description  of  the  process  will 
therefore  be  given  in  the  chapter  on  butter  analysis. 

THE  REICHERT-MEISSL  NUMBER. 

The  Reichert-Meissl  number  is  the  number  of  cubic  centimeters 
of  tenth-normal  caustic  alkali  required  to  neutralize  the  soluble 
volatile  acids  obtained  from  5  grams  of  a  fat  by  the  Reichert  dis- 
tillation process. 

This  number  serves  as  a  comparative  measure  of  the  acids  of 
low  molecular  weight.  Its  principal  use  is  in  the  examination  of 
butter  fat  and  the  detailed  description  will  be  given  in  that  con- 
nection. The  Reichert  number  is  about  one-half  the  Reichert- 
Meissl  number,  Reichert  having  originally  recommended  the 
use  of  2.5  grams  of  fat.  Among  the  common  oils  and  fats  the 
Reichert-Meissl  numbers  are  usually  less  than  i.o.  Butter  fat  has 
a  high  Reichert-Meissl  number  and  as  this  determination  is  used 
principally  in  the  examination  of  butter,  it  will  be  described  in  the 
chapter  on  butter  analysis. 

THE  IODINE  OR  HUBL  NUMBER. 

The  iodine  or  Hubl  number  is  the  percentage  of  iodine  (or  of 
iodine  chloride  or  bromide  expressed  in  terms  of  iodine)  absorbed 
by  the  sample. 

This  number  gives  a  quantitative  measure  of  the  unsaturated 
fatty  acids  (or  of  the  "  number  of  double  bonds  ")  in  a  fat  or  wax. 

Mills,  Snodgrass  and  Akitt  were  probably  the  first  to  make 
systematic  use  of  the  halogen  absorbing  power  in  fat  analysis.  In 
their  experiments*  the  oil  or  fat  to  be  tested  was  dissolved  in  car- 
bon tetrachloride  and  titrated  with  a  standard  solution  of  bromine 
in  carbon  tetrachloride  as  long  as  the  bromine  was  absorbed.  At 

*  Journ.  Soc.  Chem.  2nd.,  1883,  2,  435  ;   1884,  3,  366. 


OILS,    FATS,   AND    WAXES.  131 

about  the  same  time,  Hubl  published*  a  method  based  upon  the  use 
of  iodine  in  an  alcoholic  solution  of  mercuric  chloride  which  was  so 
carefully  worked  out  in  all  of  its  details  that  the  original  form  of 
the  process  is  still  used  in  many  laboratories  in  preference  to  any 
of  the  modifications  which  have  been  proposed.  Recently,  how- 
ever, the  Wijsf  and  the  HanusJ  modifications  have  been  largely 
used,  and  it  is  probable  that  they  will  gradually  replace  the  Hubl 
process.  These  three  methods  will  be  given  here.  The  bromine 
absorption  method  as  developed  by  Mcllhiney§  can  be  used  in  place 
of  the  iodine  methods,  but  is  more  especially  adapted  to  the  exami- 
nation of  linseed  oil  for  rosin  oil  or  rosin,  and  will  be  referred  to 
in  that  connection  in  the  next  chapter. 

Method  of  Hubl. 

Reagents. —  I.  Iodine  solution.  Dissolve  26  grams  of  pure 
iodine  in  500  c.c.  of  95  per  cent,  alcohol.  Dissolve  30  grams  of 
mercuric  chloride  in  500  c.c.  of  alcohol  of  the  same  strength.  Mix 
the  two  solutions  at  least  12  hours  before  using.  As  this  solution 
is  expensive  and  does  not  keep  well,  no  more  than  a  week's  supply 
should  be  made  at  one  time. 

2.  Standard  solution  of  sodium  thiosulphate.  Dissolve  24  grams 
of  the  crystallized  salt  in  a  liter  of  water,  allow  to  stand  at  least  24 
hours  and  then  determine  the  strength  of  the  solution  in  terms  of 
iodine.  While  any  of  the  well-known  methods  may  be  used,  it  is 
convenient  to  standardize  the  thiosulphate  solution  as  follows : 

Weight  3.8694  grams  of  pure  dry  potassium  dichromate,  dis- 
solve in  water  and  dilute  to  1000  c.c.  In  a  well-filled  tightly-stop- 
pered bottle,  this  standard  solution  of  dichromate  can  be  kept  in- 
definitely without  deterioration.  Each  c.c.  of  this  solution  is 
equivalent  to  o.oi  gram  iodine.  Mix  25  c.c.  of  a  15  per  cent, 
potassium  iodide  solution  with  5  c.c.  of  hydrochloric  acid,  add  50 
c.c.  of  the  dichromate  solution,  and  titrate  the  liberated  iodine  by 
means  of  the  thiosulphate  solution,  observing  procedure  and  pre- 
cautions described  below.  Calculate  the  amount  of  iodine  con- 
sumed by  each  c.c.  of  the  thiosulphate  solution.  As  a  precaution 
the  strength  of  either  the  dichromate  or  the  thiosulphate  solution 
should  also  be  determined  by  an  independent  method. 

*  Dingl.  polyt.  Journ.,  1884,  253,  281.     Reprinted  by  Ephraim,  loc.  cit. 

f  Ber.  deut.  cheni.  Ges.,  1898,  31,  750. 

J  ZtsJir.  Unters.  Nahr.-Genussm.,  1901,  4,  913. 

§  Journ.  Amer.  Chem.  Soc,,  1884,  16,  24.5  ;   1899,  21,  1084;   1902,  24,  1109. 


132  ORGANIC  ANALYSIS. 

3.  An  approximately  15  per  cent,  solution  of  pure  potassium 
iodide  in  cold,  recently  boiled,  distilled  water. 

4.  Freshly  prepared  starch  solution,  I  part  starch  to  200  parts  of 
water,  for  use  as  indicator. 

5.  Pure  chloroform. 

6.  Cold,  recently  boiled,  distilled  water. 

Determination.  —  Thoroughly  clean  and  dry  two  or  more  thin 
Erlenmeyer  flasks,  of  the  form  made  for  this  purpose,  having 
accurately  ground  glass  stoppers  and  flaring  mouths  which  form  a 
gutter  between  the  stopper  and  the  lip.  Into  one  of  these  flasks 
weigh  such  an  amount  of  the  sample  as  will  absorb  between  0.3 
and  0.4  gram  of  iodine  *  and  add  10  c.c.  of  chloroform.  When  the 
sample  has  completely  dissolved,  add  25  c.c.  or  30  c.c.  of  the 
mixed  iodine  solution,  stopper  carefully,  fill  the  gutter  around  the 
stopper  with  potassium  iodide  solution  to  guard  against  loss  of 
iodine,  shake  gently,  and  allow  the  flask  to  stand  in  a  cool  dark 
closet  for  3  to  20  hours  according  to  the  nature  of  the  sample.  In 
another  clean  dry  flask  of  the  same  size  and  form  make  a  blank 
determination  using  the  same  amounts  of  chloroform,  iodine  solu- 
tion, and  potassium  iodide.  Allow  the  two  flasks  to  stand  side 
by  side  for  the  same  length  of  time.  When  the  absorption  is 
complete,  lift  the  stopper  in  such  a  way  that  its  lower  surface  will 
be  washed  by  the  iodide  solution  from  the  gutter;  add  100  c.c.  of 
cold,  recently  boiled,  distilled  water  and  20  c.c.  more  of  the  potas- 
sium iodide  solution,  washing  down  the  sides  of  the  flask  with  the 
latter.  In  case  a  red  precipitate  of  mercuric  iodide  appears,  add 
more  potassium  iodide  until  the  precipitate  is  dissolved.  Titrate 
the  excess  of  iodine  at  once  by  means  of  the  standard  thiosulphate 
solution.  The  latter  may  be  run  in  rapidly  until  the  iodine  is 
nearly  consumed  and  the  solution  is  only  light  yellow ;  then  add 
2  c.c.  of  the  starch  solution  and  finish  the  titration  carefully  but 
without  delay.  As  the  end  point  is  approached,  stopper  the  flask 
quickly  after  each  addition  of  thiosulphate  and  shake  vigorously 
to  ensure  thorough  and  rapid  mixing  of  the  contents.  The  end 
point  should  be  sharper  than  in  standardizing  since  in  this  case 
there  is  no  green  color  due  to  chromium  and  the  solution  passes 
at  once  from  blue  to  nearly  colorless.  The  difference  between  the 
volume  of  thiosulphate  solution  required  for  the  blank  test  and 
that  required  for  the  solution  containing  the  sample  gives  a  meas- 

*  See  iodine  numbers  in  table  at  the  end  of  this  chapter. 


OILS,    FATS,   AND    WAXES.  133 

ure  of  the  iodine  absorbed  by  the  latter.  Calculate  the  iodine 
absorbed  in  terms  of  percentage  of  the  original  weight  of  sample. 

Notes.  —  Most  of  the  difficulties  which  are  met  in  using  this 
method  are  due  to  impure  reagents  or  failure  to  observe  carefully 
the  conditions  worked  out  by  Hubl.  Probably  the  most  important 
sources  of  error  are:  (I)  The  use  of  impure  chloroform  or  water 
containing  dissolved  air  causing  a  liberation  of  iodine  from  the 
potassium  iodide ;  (2)  loss  of  iodine  from  the  use  of  vessel  with 
imperfectly  fitting  stopper  or  from  titrating  at  too  high  tempera- 
ture or  with  too  much  exposure  to  air;  (3)  deterioration  of  the 
iodine  solutions  due  chiefly  to  impurities  in  the  alcohol  or  too 
high  temperature ;  (4)  variations  in  the  excess  of  iodine  over  that 
absorbed ;  (5)  variations  in  the  length  of  time  allowed  for  the 
reaction. 

The  small  amount  of  oil  needed  for  this  determination  can  con- 
veniently be  drawn  from  near  the  center  of  the  sample  bottle 
by  means  of  clean  glass  tubing  of  about  2  mm.  internal  diameter. 
Use  a  piece  of  thin  tubing  like  a  pipette,  wiping  it  free  from  oil  on 
the  outside  and  allowing  it  to  deliver  drop  by  drop  into  the  weighed 
flask  which  should  then  be  stoppered  and  reweighed  at  once.  If 
more  than  one  sample  of  oil  is  to  be  tested,  delays  can  be  avoided 
by  having  a  number  of  pieces  of  the  tubing  cleaned  and  dried  in 
advance  so  that  each  can  be  rejected  after  using  it  for  one  sample. 

The  best  results  are  obtained  by  using  such  proportions  of  oil 
and  of  iodine  solution  as  to  have  present  from  two  to  three  times 
the  amount  of  iodine  which  will  be  absorbed.  With  samples 
which  absorb  only  small  amounts  of  iodine  the  reaction  is  probably 
complete  in  two  or  three  hours,  but  with  linseed  and  similar  oils  it 
is  necessary  to  allow  the  iodine  solution  to  act  longer.  It  has 
been  claimed  that  several  hours  standing  introduces  an  error 
through  the  deterioration  of  the  iodine  solution  in  the  blanks,  but 
this  need  not  be  feared  if  the  reagents  are  pure  and  the  flasks 
tightly  stoppered  and  kept  in  a  cool  dark  place.  Unless  it  is  cer- 
tain that  a  shorter  time  will  be  sufficient  it  is  well  to  allow  the 
determinations  to  stand  over  night.  The  form  of  vessel  to  be  used 
for  the  test  is,  of  course,  immaterial  so  long  as  loss  of  iodine  is 
avoided.  Some  prefer  to  weigh  the  sample  on  a  small  watch  glass 
and  place  the  latter  with  the  sample  in  a  wide-mouth  glass  stop- 
pered bottle.  A  blank  test  must  be  made  with  each  determination, 
or  if  several  samples  are  treated  at  once  there  should  be  at  least 
two  or  three  blanks. 


134  ORGANIC  ANALYSIS. 

For  discussion  of  the  theory  of  the  action  of  Hiibl's  solution 
see  Levvkowitsch's  Oils,  Fats,  and  Waxes,  or  Gill's  Oil  Analysis. 

Method  of  Wijs. 

Wijs  found  that  a  solution  of  iodine  monochloride  in  glacial 
acetic  acid  acts  in  the  same  manner  as  Hiibl's  solution,  but  more 
quickly.  Moreover,  the  reagent  is  much  more  stable  than  that  of 
Hlibl,  so  that  the  same  solution  can  be  used  for  several  months  and 
blank  tests  are  not  so  frequently  required. 

Reagents. —  i.  Glacial  acetic  acid.  This  must  contain  not  over 
0.5  per  cent,  of  water  and  no  impurity  capable  of  reducing  potas- 
sium dichromate.  Test  by  adding  a  small  amount  of  a  solution  of 
dichromate  in  sulphuric  acid  and  warming  on  a  water-bath ;  no 
green  tinge  should  appear. 

2.  Iodine  chloride   solution.      Dissolve    12.5    to    13    grams   of 
iodine  in  a  liter  of  the  glacial  acetic  acid,  warming  gently  to  aid 
solution,  if   necessary.     Cool,  withdraw    25   c.c.,  add    potassium 
iodide  solution,  and  determine  the  iodine  content  by  titration  with 
standard  thiosulphate ;  into  the  remainder  of  the  solution  pass  a 
current  of  chlorine  until  the  deep  iodine  color  changes  to  an  orange 
yellow,  showing  that  the   conversion  of  iodine  to  iodine  mono- 
chloride  is  complete.     A  portion  of  the  solution  titrated  with  thio- 
sulphate should  now  show  twice  the  original  halogen  content.     In 
case  an  excess  of  chlorine  is  found,  a  weighed  amount  of  iodine 
equivalent  to  this  excess  should  be  dissolved  in  the  solution. 

3.  Standard  solution  of  sodium  thiosulphate,  as   in  the  method 
of  Hubl. 

4.  Pure  chloroform,  potassium   iodide,  starch  solution,  and  dis- 
tilled water  as  described  under  Hiibl's  method. 

Determination. —  The  determination  is  carried  out  in  exactly  the 
manner  described  for  the  Hubl  method  except  that  10  c.c.  of  the 
potassium  iodide  solution  are  sufficient  and  a  much  shorter  time 
is  required  for  the  reaction.  With  non-drying  oils  the  addition  of 
iodine  chloride  is  complete  in  15  minutes.  Ordinarily  the  solu- 
tion should  be  titrated  after  standing  one  hour. 

Notes. — As  the  solution  of  iodine  chloride  in  acetic  acid  is  more 
viscous  and  has  a  higher  coefficient  of  expansion  than  ordinary 
aqueous  solutions,  the  portions  added  must  be  measured  as  carefully 
as  possible,  allowing  a  uniform  time  for  the  burette  to  drain,  and 
observing  that  the  temperature  does  not  vary  more  than  three  or 


OILS,   FATS,   AND    WAXES.  135 

four  degrees.  With  these  precautions  it  is  not  always  necessary  to 
make  a  blank  test  with  each  determination  after  a  sufficient  number 
of  such  tests  have  been  made  to  show  that  the  solution  is  not 
changing  appreciably  in  strength. 

Method  of  Hanus. 

Hanus  suggested  that  Wijs'  method  be  modified  by  the  use  of 
iodine  bromide  in  place  of  iodine  chloride.  As  an  excess  of  iodine 
does  no  harm,  the  preparation  of  the  reagent  is  simpler  than  in 
the  preceding  method. 

The  reagents  required  are  the  same  as  for  the  Wijs  method  ex- 
cept that  the  iodine  bromide  solution  is  prepared  by  dissolving 
1 3.2  grams  of  iodine  and  3  c.c.  of  bromine  in  a  liter  of  glacial  acetic 
acid.  The  iodine  may  be  dissolved  first  in  the  greater  part  of  the 
acid,  heating  to  aid  solution  ;  the  bromine  is  added  after  cooling  the 
solution  and  the  volume  then  brought  to  a  liter. 

The  manipulation  is  the  same  as  in  the  Wijs  method,  but  as  the 
reaction  is  not  quite  so  rapid  an  hour  should  be  allowed  in  all 
cases.  It  is  well  to  make  the  determination  in  duplicate,  allowing 
more  time  in  one  case  than  in  the  other.  After  adding  the  potas- 
sium iodide  solution  shake  thoroughly  before  adding  water. 

Comparison  of  the  Hubl^  Wijs,  and  Hanus  Methods. 

Wijs  based  his  modification  on  the  belief  that  iodine  chloride  is 
the  active  substance  in  Hubl's  solution  and  that  the  more  stable 
solution  of  iodine  chloride  in  acetic  acid  should  give  the  same 
theoretical  results  with  greater  certainty.  That  higher  results  were 
often  obtained  by  his  method  Wijs  attributed  to  the  deterioration 
or  incomplete  action  of  the  Hubl  solution  as  ordinarily  prepared. 
Lewkowitsch  *  confirms  this,  stating  that  when  the  Hubl  method 
is  carried  out  under  the  best  conditions  the  results  of  the  two 
methods  agree. 

Tolman  and  Munson,f  however,  in  an  extended  comparison  of 
the  three  methods  almost  invariably  obtained  higher  results  by  the 
Wijs  than  by  the  Hubl  method.  Hanus'  method  gave  intermediate 
results,  usually  agreeing  quite  closely  with  the  Hubl  numbers. 
Similar  results  on  a  smaller  number  of  samples  had  previously 
been  published  by  HuntJ  and  have  since  been  obtained  by  W. 

*  Analyst,  1899,  24,  259. 

•\Journ.  Amer.  Chem.  Soc.,  1903,  25,  244. 

%Journ.  Soc.  Chem.  Ind.,  1902,  21,  454. 


136  ORGANIC  ANALYSIS. 

G.  Tice.  *  For  oils  having  iodine  numbers  below  100,  the  three 
methods  can  be  used  interchangeably  as  the  differences  in  results 
will  rarely  exceed  1.5  units  which  is  only  about  one-tenth  of  the 
variation  which  occurs  among  pure  oils  of  the  same  species.  In 
applying  the  Wijs  solution  to  oils  having  much  higher  iodine  num- 
bers it  is  to  be  remembered  that  results  as  much  as  10  units  in  ex- 
cess of  these  given  by  Hubl's  solution  will  sometimes  be  obtained. 

The  method  of  Hubl  is  "  official  "  in  the  United  States  but  that 
of  Hanus  has  been  adopted  by  the  Association  of  Official  Agri- 
cultural Chemists  as  a  provisional  optional  method  for  the  exami- 
nation of  edible  oils  and  fats.  The  method  of  Wijs  is  more  com- 
monly used  in  England  and  is  recommended  in  preference  to  the 
Hanus  method  by  Lewkowitsch  f  and  Archbutt,  J  the  latter  point- 
ing out  especially  that  the  Wijs  solution  gives  approximately  the 
theoretical  iodine  number  for  turpentine  (which  is  often  mixed 
with  fatty  oils)  while  that  of  Hanus  gives  much  lower  results. 

In  comparing  analytical  results  with  the  numbers  found  in 
"  tables  of  constants  "  it  should  be  remembered  that  the  latter  are 
based  in  part  upon  results  obtained  (not  always  under  the  best 
conditions)  by  Hubl's  method,  and  in  part  upon  later  results  de- 
termined according  to  Wijs.  Such  tables  tend  to  show  a  greater 
range  than  would  be  found  by  the  use  of  either  method  alone, 
this  being  especially  true  of  the  oils  having  high  iodine  numbers. 

MAUMENE  NUMBER  —  SPECIFIC  TEMPERATURE  REACTION. 
Long  before  the  introduction  of  any  of  the  halogen  absorption 
methods  Maumene  §  tested  olive  oil  for  adulterants  such  as  poppy- 
seed  oil  by  mixing  fixed  volumes  of  oil  with  strong  sulphuric  acid 
and  observing  the  rise  in  temperature  which  is  much  greater  with 
seed  oils  than  with  olive.  The  rise  of  temperature,  expressed  in 
degrees  C.,  which  occurs  on  mixing  10  c.c.  of  strong  sulphuric 
acid  with  50  grams  of  oil  is  commonly  known  as  the  Maumene 
number.  Thomson  and  Ballantyne  ||  pointed  out  the  discrep- 
ancies which  may  be  introduced  through  variations  in  the  strength 
of  the  acid  used,  and  showed  that  they  can  be  largely  avoided 
by  comparing  the  rise  observed  in  the  case  of  an  oil  with  that 

*  In  the  Havemeyer  Laboratory,  Columbia  University. 

fOils,  Fats  and  Waxes  (3d  Ed.),  I.,  252. 

\Journ.  Soc.  Chem.  Ind.,  1904,  23,  306. 

§  Compt.  rend.,  1852,  35,  572. 

\\Journ.  Soc.  Chem.  Ind.t  1891,  10,  234. 


OILS,   FATS,   AND    WAXES.  137 

shown  by  water  under  the  same  conditions,  the  latter  being 
taken  as  100.  The  result  thus  obtained  is  known  as  the  specific 
temperature  reaction  or  specific  Maumene  number.  Thus  if  50 
grams  of  water  mixed  with  10  c.c.  of  sulphuric  acid  showed  a  rise 
of  40°  and  50  grams  of  an  oil  showed  under  the  same  conditions 
a  rise  of  36°  the  specific  Maumene  number  of  the  oil  would  be  90. 

With  fresh  fatty  oils  the  rise  of  temperature  depends  mainly 
upon  the  presence  of  glycerides  of  the  unsaturated  acids  and  the 
Maumene  and  Hubl  numbers  are  therefore  roughly  proportional. 
This  relation,  however,  is  not  sufficient  to  permit  of  the  determi- 
nation of  the  Maumene  in  place  of  the  iodine  number  except  for 
rough  work  or  as  a  preliminary  test.  The  greatest  value  of  this 
temperature  reaction  lies  in  the  fact  that  as  oils  become  altered  by 
absorption  of  oxygen  from  the  air,  the  Maumene  numbers  increase 
while  the  iodine  numbers  decrease.  The  significance  of  this  rela- 
tion is  more  fully  explained  in  the  next  chapter. 

Apparatus  and  Reagents.  —  A  deep  beaker  of  150  to  200  c.c. 
capacity  should  be  used  for  this  test  and  should  be  so  jacketed  as 
to  prevent  a  rapid  loss  of  heat.  This  is  done  by  placing  it  in  a 
larger  beaker  or  a  metal  cup  and  filling  the  space  with  asbestos  or 
other  dry  porous  material.  If  a  new  nest  of  beakers  is  at  hand 
the  test  can  be  made  in  one  of  the  smaller  beakers  using  the  re- 
mainder of  the  nest  with  the  packing  intact  as  a  jacket.  The 
insulation  should  be  sufficient  to  prevent  the  outer  vessel  from  be- 
coming perceptibly  warm  when  a  test  is  made.  The  only  reagent 
required  is  strong  sulphuric  acid  which  can  be  added  either  from  a 
burette  or  (more  satisfactorily)  from  a  10  c.c.  pipette.  For  observ- 
ing the  rise  in  temperature  use  a  thermometer  graduated  in 
degrees  on  which  the  readings  can  be  taken  with  accuracy  to  0.2 
degree. 

Determination.  —  The  oils  to  be  tested,  the  acid  and  apparatus  to 
be  used,  and  the  distilled  water  which  is  to  serve  for  comparison 
must  all  have  the  same  initial  temperature,  which  should  be 
between  20°  and  24°.  This  will  usually  be  the  case  if  they  have 
stood  side  by  side  at  room  temperature  for  several  hours.  Measure 
50  c.c.  of  water  into  the  beaker,  introduce  the  thermometer  and 
note  the  temperature;  add  IO  c.c.  of  the  acid,  stirring  thoroughly 
with  the  thermometer  so  that  the  solution  is  kept  well  mixed  and 
the  temperature  rises  steadily.  At  intervals  of  a  few  seconds  bring 
the  bulb  of  the  themometer  to  the  center  of  the  solution  and 


138  ORGANIC  ANALYSIS. 

observe  the  temperature.  Record  the  highest  reading  found. 
When  the  mixture  in  the  beaker  has  reached  its  maximum  tem- 
perature it  should  be  rejected  at  once  to  avoid  unnecessary  warm- 
ing of  the  apparatus,  since  the  initial  temperature  must  be  restored 
before  beginning  another  test.  After  the  rise  with  water  has  been 
found  by  duplicate  determinations  which  do  not  differ  by  more 
than  0.4°,  dry  the  beaker  thoroughly,  weigh  into  it  50  grams  of 
oil  (within  0.05  gram),  and  treat  with  10  c.c.  of  acid  in  exactly  the 
same  manner.  The  mixture  often  becomes  very  viscous,  requir- 
ing vigorous  stirring  to  ensure  a  uniform" temperature.  In  report- 
ing results  give  the  actual  rise  observed  with  oil  and  with  water 
as  well  as  the  specific  temperature  reaction. 

Notes.  —  By  the  use  of  a  pipette  having  a  fine  outlet  requiring 
30  to  60  seconds  for  the  10  c.c.  of  acid  to  flow  into  the  beaker, 
more  thorough  mixing  becomes  possible  and  better  results  are  se- 
cured. In  order  to  avoid  trouble  in  cleaning  the  beaker  it  should 
be  emptied  and  wiped  with  dry  cotton  waste  or  porous  paper  while 
still  warm.  The  acid  used  must  be  protected  from  unnecessary 
exposure  to  air  as  it  readily  absorbs  moisture  to  an  extent  suffi- 
cient to  cause  an  appreciably  lower  temperature  reaction.  In 
testing  edible  and  lubricating  oils  the  strongest  available  sulphuric 
acid  should  be  used.  In  the  case  of  drying  oils  and  fish  or  other 
marine  animal  oils,  the  addition  of  such  strong  acid  to  the  undi- 
luted oil  causes  violent  frothing  and  decomposition.  This  maybe 
avoided  *  by  the  use  of  sulphuric  acid  of  such  strength  as  to  give 
a  rise  of  35°  when  added  to  water  under  the  conditions  to  be  used 
in  testing  the  oil.  An  important  modification  of  this  test  has  been 
proposed  by  MitchelLf 

THE  ACETYL  NUMBER. 

The  acetyl  number  is  the  number  of  milligrams  of  potassium 
hydroxide  required  to  neutralize  the  acetic  acid  obtained  by  saponi- 
fication  of  one  gram  of  the  acetylated  fat  or  wax. 

It  indicates  the  proportion  of  alcoholic  hydroxyl  groups  in  the 
original  substance.  If  the  latter  consists  essentially  of  triglycerides, 
the  acetyl  number  serves  as  a  measure  of  the  hydroxy-acids  pres- 
ent. Free  alcohols,  if  present,  increase  the  acetyl  numbers.  Lew- 

*  Sherman,  Danziger  and  Kohnstamm  :  Journ.  Amer.  Chem.  Soc.,  1902,  24,  266. 
f  Analyst,  1901,  26,  169.     See  also  Sherman  and  Falk  :  Journ.  Amer.  Chem.  Sot., 
1903,  25,  7I3- 


OILS,    FATS,   AND    WAXES.  139 

kowitsch  finds  that  free  fatty  acids  of  the  stearic  series  may  also 
react  in  such  a  way  as  to  increase  the  apparent  acetyl  numbers, 
and  therefore  prefers  to  acetylate  the  original  fat  or  wax  rather 
than  to  work  with  the  mixed  fatty  acids  as  was  previously  recom- 
mended by  Benedikt  and  Ulzer.  * 

The  method  of  Lewkowitsch  is  essentially  as  follows :  f 

Boil  10  grams  of  the  substance  with  twice  its  weight  of  acetic 
anhydride  in  a  round  bottomed  flask  under  a  reflux  condenser  for 
two  hours;  pour  the  resulting  mixture  into  a  large  beaker  contain- 
ing 500  c.c.  of  hot  water  and  boil  for  half  an  hour,  passing  a  slow 
current  of  carbon  dioxide  through  the  solution  to  prevent  bump- 
ing. Allow  the  mixture  to  separate  into  two  layers,  siphon  off  the 
water,  and  boil  the  oily  layer  with  three  successive  portions  of  fresh 
water,  the  last  of  which  should  not  react  acid  to  litmus  paper. 

All  free  acetic  acid  having  been  removed,  the  acetylated  fat  is 
carefully  separated  from  water  and  further  dried  by  filtering  through 
anhydrous  paper  in  a  drying  oven. 

Weigh  2  to  5  grams  of  the  acetylated  fat  and  saponify  with  a 
measured  volume  of  standard  alcoholic  potash  as  in  the  determina- 
tion of  the  saponification  number ;  evaporate  nearly  to  dryness  to 
expel  the  alcohol,  dissolve  the  soap  in  water  and  add  an  amount  of 
standard  sulphuric  acid  exactly  equivalent  to  the  alkali  used  for 
saponification.  Warm  gently  until  the  fatty  acids  separate  as  a 
layer  at  the  top.  Filter  through  wet  paper,  wash  the  fatty  acids 
with  boiling  water  until  the  filtrate  is  no  longer  acid,  and  titrate 
the  filtrate  and  washings  with  tenth-normal  alkali,  using  phenol- 
phthalein  as  indicator.  Deduct  the  amount  of  alkali  required  to  neu- 
tralize any  soluble  fatty  acids  in  the  original  substance  and  calculate 
the  acetic  acid  found,  in  terms  of  acetyl  number  as  defined  above. 

Instead  of  filtering  and  washing  with  water,  the  acetic  acid  can 
be  separated  from  the  fatty  acids  by  distilling  with  steam,  but  this 
requires  considerably  more  time  than  the  filtration  process. 

In  the  examination  of  waxes  the  acetyl  numbers  show  the  pro- 
portional amounts  of  free  alcohols.  Fats  and  fatty  oils  contain 
free  alcohols  (cholesterol,  phytosterol,  etc.),  but  in  very  small 
amounts  only,  so  that  the  acetyl  numbers  are  low  except  in  oils 
which  contain  hydroxy-acids.  (Chapter  VIII.)  The  acetyl  num- 
ber of  castor  oil  is  about  150.  Other  oils  rarely  show  acetyl 

*  Monatsh.    Chem.%  1887,  8,  40. 

•fjourn.  Soc.  Chem.  Ind.,  1897,  16,  503;  Oils,  Fats,  and  Waxes  (3d  ed.),  268. 


ORGANIC  ANALYSIS. 

numbers  higher  than  10,  unless  they  have  been  exposed  to  atmos- 
pheric oxidation,  in  which  case  hydroxy-acids  may  have  been 
formed  from  the  unsaturated  acids  originally  present. 

SPECIFIC  GRAVITY. 

The  specific  gravities  of  the  glycerides  depend  upon  the  fatty 
acids  which  they  contain.  Comparing  acids  of  the  same  homol- 
ogous series  the  specific  gravity  was  found  to  decrease  with  in- 
creasing molecular  weight ;  while  between  corresponding  acids  of 
different  series  it  increases  with  the  number  of  double  bonds  and 
of  hydroxyl  groups. 

Fresh  specimens  of  the  same  kind  vary  but  little  in  specific 
gravity,  and  the  determination  of  this  property  is  often  useful  in 
differentiating  oils,  fats,  and  waxes  and  in  testing  them  for  adultera- 
tions. The  determination  can  be  made  either  by  means  of  a  very 
delicate  special  hydrometer,  by  the  Westphal  balance,  or  by  one 
of  the  methods  described  in  Chapter  III.  On  account  of  the  high 
coefficient  of  expansion  of  oils  it  is  important  that  the  temperature 
at  which  the  specific  gravity  is  taken  be  accurately  known.  This 
is  conveniently  accomplished  by  using  the  thermometer  sinker  with 
the  Westphal  balance. 

Directions  for  the  use  of  the  Westphal  balance  are  usually  sup- 
plied with  the  instrument.  It  should  always  be  placed  upon  a 
firm  level  table  and  very  carefully  adjusted  before  using.  In  setting 
up  the  instrument  be  sure  that  the  leveling  screw  is  directly  be- 
neath the  arm  which  supports  the  beam.  Place  the  latter  in  posi- 
tion and  hang  the  sinker.  Compare  the  height  of  the  beam  with 
that  of  the  cylinder  which  is  to  contain  the  oil  and  lengthen  or 
shorten  the  standard  if  necessary,  then  carefully  adjust  the  balance 
by  means  of  the  leveling  screw  until  the  point  projecting  from  the 
end  of  the  beam  stands  exactly  opposite  the  fixed  point  at  the 
left.  Nearly  fill  the  cylinder  with  the  oil  at  about  14°.  Lift  the 
sinker,  place  the  cylinder  under  the  end  of  the  beam  and  replace 
the  sinker  so  that  it  hangs  freely  in  the  oil.  The  specific  gravity 
is  now  found  by  placing  weights  on  the  beam  until  the  balance  is 
restored.  Make  the  final  adjustment  of  the  smallest  weight  when 
the  thermometer  in  the  sinker  shows  the  desired  temperature  — 
for  oils,  15.5°.  The  specific  gravity  is  read  directly  from  the  posi- 
tion of  the  weights  on  the  beam.  If  the  largest  weight  is  at 
9,  the  second  at  3,  the  third  at  I,  and  the  fourth  at  5,  the  spe- 


OILS,    FATS,   AND    WAXES.  141 

cific  gravity  of  the  oil  is  0.9315.  The  readings  should  be  made 
to  four  decimal  places  (i.  e.,  all  four  sizes  of  weights  should  be 
used)  and  the  error  of  determination  should  not  exceed  0.0005. 
By  using  the  same  sinker  on  the  analytical  balance  or  by  weighing 
the  oil  in  a  pyknometer,  more  accurate  results  can  be  obtained. 
The  Westphal  balance  is,  however,  sufficiently  delicate  for  routine 
work  if  carefully  used.  Care  must  be  taken  that  the  sinker  is 
always  suspended  at  the  same  depth  in  the  liquid  under  examina- 
tion. The  graduation  of  the  beam  should  be  tested  to  see  that 
the  spaces  are  all  of  equal  length,  and  the  results  should  occasionally 
be  verified  by  comparative  determinations  with  the  pyknometer. 
In  working  with  very  viscous  oils  it  is  advantageous  to  use  a  sinker 
of  high  specific  gravity.* 

For  the  determination  of  specific  gravity  at  temperatures  con- 
siderably above  that  of  the  laboratory  it  is  convenient  to  use  a 
Sprengel  or  Ostwald  pyknometer.  This  is  filled  with  the  sample, 
suspended  in  water  at  the  required  temperature  and  adjusted,  then 
removed,  dried  on  the  outside,  cooled  to  room  temperature  and 
weighed.  For  a  detailed  description  of  the  standardization  of 
pyknometers  at  high  temperatures  consult  any  standard  work  on 
physico-chemical  measurements  or  Bulletin  46  or  65,  Bureau  of 
Chemistry,  U.  S.  Department  of  Agriculture. 

INDEX  OF  REFRACTION. 

The  index  of  refraction,  like  the  specific  gravity,  increases  with 
the  proportion  of  unsaturated,  or  of  hydroxy-acids.  Unlike  the 
specific  gravity,  the  index  of  refraction  increases  with  the  molec- 
ular weight  in  the  homologous  series  of  saturated  acids  so  that 
the  presence  of  the  fatty  acids  of  low  molecular  weight  in  butter 
causes  this  fat  to  have  a  lower  index  of  refraction  than  other  animal 
fats,  while  its  specific  gravity  is  higher.  In  almost  all  of  the  fatty 
oils  the  index  of  refraction  varies  with  the  specific  gravity  so  that 
in  routine  work  it  is  not  necessary  to  make  both  determinations 
but  either  may  be  used  to  confirm  the  inferences  drawn  from  the 
other  determinations/)- 

The  index  of  refraction  has  usually  been  determined  by  means 
of  the  Abbe  or  the  Pulfrich  refractometer.  The  "  oleo-refractome- 

*McGill:  Analyst,  1896,  21,  156. 

f  For  a  very  full  discussion  of  the  relation  of  index  of  refraction  to  specific  gravity 
in  oils  and  fats  see  Proctor  :  Jonrn.  Soc.  Chem.  Ind.,  1898,  17,  1021. 


142 


ORGANIC  ANALYSIS. 


ter  "  of  Amagat  and  Jean  is  a  differential  instrument  in  which  the 
results  are  found  in  terms  of  an  arbitrary  scale,  the  value  of 
which,  according  to  Allen,  is  not  constant.  The  readings  of  this 
instrument  cannot,  therefore,  be  compared  with  determinations  of 
the  true  index  of  refraction.  The  "  butyro-refractometer  "  of  Zeiss 
also  has  an  an  arbitrary  scale  but  in  this  case  the  values  of  the 
scale  readings  are  known  and  can  be  compared  with  direct  deter- 
minations of  the  index  of  refraction  as  follows  : 


Butyro-refrac- 
tometer 
'Reading. 

Index  of  Re- 
fracticn. 

Butyro-refrac- 
tometer 
Reading. 

Index  of  Re- 
fraction. 

Butyro-refrac- 
tometer 
Reading. 

Index  of  Re- 
fraction. 

O 
10 
20 
30 

I.422O 
1.4300 

1-4377 
1.4452 

40 

5° 
60 
70 

1.4524 

1.4593 

1.4659 
1.4723 

80 
90 
IOO 

1.4783 
1.4840 
1.4895 

Since  the  butyro-refractometer  is  constructed  to  cover  only  such 
a  range  in  the  index  of  refraction  as  is  likely  to  be  met  in  work 
with  fats  and  oils,  it  is  possible  to  make  more  delicate  readings 
with  this  instrument  than  with  the  Abbe  refractometer.  It  is  pref- 
erable, however,  to  express  all  results  in  terms  of  the  index  of  re- 
fraction, especially  as  some  substances  frequently  mixed  with  oils 
and  fats  are  too  highly  refractive  to  be  examined  in  the  butyro- 
refractometer. 

The  index  of  refraction  decreases  with  rising  temperature.  The 
rate  of  change  is  nearly  constant  for  the  common  oils  and  fats,  a 
rise  of  i°  causing  a  diminution  of  0.000365  in  the  index  of  re- 
fraction.* 

Directions  for  the  determination  of  the  index  of  refraction  will 
be  found  in  Kohlrausch's  Physical  Measurements,  Leach's  Food 
Inspection  and  Analysis,  Lewkowitsch's  Oils,  Fats,  and  Waxes,  or 
Vaubel's  Quantitative  Bestimmung  organische  Verbindungen. 

MELTING  AND  SOLIDIFYING  POINTS  —  TITER  TEST. 

The  melting  point  of  a  fat,  or  of  the  mixed  fatty  acids  obtained 
from  it,  increases  with  the  mean  molecular  weight  among  acids  of 
the  saturated  series,  while  in  mixtures  of  acids  of  the  saturated 
and  unsaturated  series  (or  their  glycerides)  the  melting  point  be- 
comes lower  as  the  proportion  of  unsaturated  compounds  increases. 


*  Proctor  :  Journ.  Soc.  Chem.  Ind. , 
Amer.  Chem.  Soc.,  1902,  24,  754. 


,  17,  1023.     Tolman  and  Munson  :  Journ. 


OILS,    FATS,   AND    WAXES.  143 

The  melting  point  of  a  solid  fat  is  best  determined  by  heating  a 
thin  disc  of  the  substance  suspended  in  a  mixture  of  water  and 
alcohol  as  described  in  Chapter  XI.  This  method  being  appli- 
cable only  to  substances  practically  insoluble  in  alcohol,  the  melt- 
ing point  of  a  mixture  of  fatty  acids  or  of  a  wax  may  be  taken  as 
follows :  * 

Draw  up  the  melted  fatty  acids  (or  the  wax)  into  a  very  thin- 
walled  capillary  tube  3  to  5  cm.  long  according  to  the  length  of 
the  bulb  of  the  thermometer  to  be  used.  Seal  one  end  of  the 
tube  and  allow  the  substance  to  cool  on  ice  for  12  to  15  hours. 
Attach  to  the  bulb  of  a  delicate  thermometer  graduated  to  0.2°, 
immerse  in  water  and  heat  very  slowly.  The  temperature  at 
which  the  substance  becomes  transparent  is  taken  as  the  melting 
point. 

The  so-called  "titer  test"  is  the  determination  of  the  solidify- 
ing point  of  the  mixed  fatty  acids.  This  test  is  largely  used  as  a 
means  of  determining  the  suitability  of  fats  for  soapmaking  and 
the  details  of  manipulation  are  now  being  studied  with  a  view  to 
the  adoption  of  a  uniform  method.f  The  Dalican  process  which 
has  been  quite  commonly  used  is  fully  described  by  Lewkowitsch. 

VISCOSITY. 

The  presence  of  glycerides  of  hydroxy-acids,  whether  natural  as 
in  castor  oil  or  artificial  as  in  the  "  blown  "  oils  of  commerce,  gives 
a  very  high  viscosity  compared  with  fatty  oils  in  which  hydroxy- 
acids  are  absent.  Among  the  latter,  viscosities  vary  as  a  rule  with 
the  melting  points  of  the  mixed  fatty  acids.  While  the  viscosity 
may  aid  in  the  identification  of  fats  and  oils  in  certain  cases,  its 
determination  is  especially  important  in  the  examination  of  lubri- 
cating oils  and  will  therefore  be  discussed  in  that  connection. 

HEAT  OF  COMBUSTION. 

The  heat  of  combustion  of  fats  and  fatty  oils  is  a  property  as 
nearly  constant  as  the  specific  gravity  to  which  it  stands  in  approx- 
imately inverse  proportion,  being  lowered  by  the  presence  of  acids 
of  low  molecular  weight,  highly  unsaturated  acids,  or  acids  con- 
taining hydroxyl  groups.  The  heats  of  combustion  of  butter-fat 
drying  oils,  castor  oil  and  blown  oils  are  therefore  lower  than 

*U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  65. 
fTolman  :  Bui.  81,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


144  ORGANIC  ANALYSIS. 

those  of  body  fats  and  such  fatty  oils  as  olive,  almond,  peanut  or  rape- 
seed.  Waxes  and  hydrocarbons  have  higher  heats  of  combustion 
than  the  glycerides.  The  same  is  true  of  the  alcohols  of  very  high 
molecular  weight  such  as  cholesterol  and  phytosterol  of  which  the 
so-called  unsaponifiable  matter  of  fats  is  chiefly  composed.  With 
a  suitable  oxygen  calorimeter  the  heat  of  combustion  can  be  very 
accurately  determined  and  the  results  are  often  useful  in  verifying 
the  conclusions  drawn  from  other  determinations.  For  fuller  dis- 
cussions see  the  Journal  of  the  American  Chemical  Society,  1896,  18, 
178;  1901,  23,  164;  1902,  24,  348;  1903,  25,  659. 

ALCOHOLS  OF  FATS  AND  WAXES. 
(UNSAPONIFIABLE  MATTER.) 

Under  the  term  "  unsaponifiable  matter"  are  included  all  sub- 
stances found  in  fats  and  waxes  which  are  insoluble  in  water  and 
do  not  combine  with  caustic  alkali  to  form  soluble  soaps.  In  a 
commercially  pure  fat  or  wax  the  "  unsaponifiable  matter  "  consists 
chiefly  of  one  or  more  alcohols  of  high  molecular  weight,  the  more 
important  of  which  are  mentioned  below. 

The  common  waxes  yield  50  to  55  per  cent,  of  insoluble  alco- 
hols while  the  total  amount  of  unsaponifiable  matter  in  fats  is 
much  lower,  being  usually  less  than  I  per  cent,  in  animal  and  less 
than  2  per  cent,  in  vegetable  fats. 

ALCOHOLS  OF  THE  SERIES  CnH2n+aO. 

Cetyl  alcohol,  C16H34O,  occurs  as  palmitate  in  spermaceti.  It  is 
a  tasteless  and  odorless  solid,  insoluble  in  water  but  soluble  in 
alcohol  and  very  easily  soluble  in  ether  or  benzene.  Melting  point 
50°;  specific  gravity  at  99°,  0.7837. 

Octodecyl  alcohol,  ClgH38O,  also  occurs  as  ester  in  spermaceti. 
It  is  similar  in  properties  to  cetyl  alcohol.  Melting  point  59° ; 
specific  gravity  at  99°,  0.7849. 

Myricyl  alcohol,  C30H62O,  occurs  as  palmitate  in  beeswax  and 
both  free  and  combined  in  carnaiiba  wax.  It  melts  at  85°  to  88° 
and  is  nearly  insoluble  in  cold,  but  readily  soluble  in  hot  alcohol. 

Other  alcohols  of  this  series  are  known  but  need  not  be  consid- 
ered here.  From  the  examples  given  it  will  be  seen  that  the  melt- 
ing points  rise  and  the  alcohols  become  less  soluble  with  increas- 
ing molecular  weight.  The  specific  gravity,  however,  does  not 
decrease  as  in  the  case  of  homologous  fatty  acids. 


OILS,   FATS,   AND    WAXES.  145 

CHOLESTEROL  AND  RELATED  ALCOHOLS. 

Cholesterol,  C26H44O,  is  the  characteristic  constituent  of  the  un- 
saponifiable  matter  of  animal  fats.  It  is  only  sparingly  soluble  in 
cold  dilute  alcohol  or  cold  petroleum  ether,  but  dissolves  readily  in 
ether,  chloroform,  carbon  bisulphide,  fatty  oils,  turpentine,  or  hot 
"strong  alcohol. 

Pkytosterol  and  sitosterol  are  alcohols,  probably  isomeric  with 
cholesterol  and  resembling  it  closely  in  physical  properties,  which 
occur  in  vegetable  fats,  the  latter  being  found  especially  in  the  oils 
of  the  cereal  grains.  The  identification  of  one  of  these  alcohols 
may  therefore  aid  in  determining  the  origin  of  an  oil  or  fat  or  in 
the  detection  of  vegetable  fats  present  as  adulterants  in  the  more 
expensive  fats  of  animal  origin.  The  identification  is  best  accom- 
plished by  converting  the  alcohol  into  an  ester,  the  properties  of 
the  esters  being  more  distinctive  than  those  of  the  free  alcohols. 
For  further  information  regarding  these  alcohols  see  Lewkowitsch's 
Oils,  Fats,  and  Waxes  (3d  Ed.),  pages  138-144  and  the  papers  of 
Bomer,*  Burian,t  Ritter,^  and  Gill  and  Tufts.  § 

REFERENCE  BOOKS. 

Alder- Wright  and  Mitchell :  Animal  and  Vegetable  Fixed  Oils, 
Fats,  Butters,  and  Waxes,  London,  1903. 

Allen :  Commercial  Organic  Analysis,  Vol.  II.,  Part  I.,  Phila- 
delphia, 1898. 

Benedikt  and  Ulzer :  Analyse  der  Fette  und  Wachsarten,  Ber- 
lin, 1897. 

Gill :  Short  Handbook  of  Oil  Analysis,  Philadelphia,  1903. 

Hopkins  :  Oil-Chemists'  Handbook,  New  York,  1900. 

Leach  :  Food  Inspection  and  Analysis,  New  York,  1904. 

Lewkowitsch  :  Chemical  Technology  and  Analysis  of  Oils,  Fats, 
and  Waxes,  London  and  New  York,  1904. 

Lunge :  Chemisch  Technische  Untersuchungsmethoden,  Bd. 
III.,  Berlin,  1900. 

Wiley :  Agricultural  Analysis,  Vol.  III.,  Easton,  1897. 

Wright :  Analysis  of  Oils  and  Allied  Substances,  London  and 
New  York,  1903. 

*  Ztschr.  Unters.  Nahr.-Genussm.,  1898,  i,  21,  8l,  532  ;  1899,  2,  46,  705  ;  1901, 
4,  865,  1070  ;  1902,  5,  1018. 

•\Monatsh.  Chem.,  1897.  18,   551. 

J  Ztschr.  physiol.  Chem.,  1902,  34,  430,  461  ;  35,  550. 

§Journ.  Amer.  Chem.  Soc.,  1903,  25,   251,  254. 


146 


ORGANIC  ANALYSIS. 


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CHAPTER  X. 
Fatty  Oils  —  Special  Methods. 

SALAD  OILS. 

Most  salad  oils  are  sold  as  olive  oil.  The  principal  substitutes 
and  adulterants  are  cotton  seed,  arachis  (peanut),  sesame,  rape, 
maize,  poppyseed,  and  lard  oils.  Both  quantitative  and  qualitative 
methods  must  be  used  in  any  thorough  examination  of  an  oil  for 
adulterants.  As  a  rule  an  oil  should  be  pronounced  adulterated 
only  when  quantitative  determinations  yield  results  which  could 
not  be  obtained  from  a  pure  oil  of  normal  character.  Qualitative 
tests  are  usually  required  to  show  which  of  several  possible  adul- 
terants is  present.  As  yet  certain  color  reactions  are  indispen- 
sable for  this  purpose,  but  in  using  these  tests  and  interpreting  the 
results  it  must  be  remembered  that  they  depend  upon  the  presence 
of  constituents  which  may  be  removed  or  destroyed  by  improved 
methods  of  refining,  and  that  olive  oil  which  has  been  altered  by 
long  exposure  to  air  in  loosely  stoppered  or  partially  filled  vessels, 
or  oil  containing  a  small  amount  of  some  accidental  impurity,  may 
give  a  reaction  which  cannot  be  distinguished  from  that  of  the 
adulterant  sought.  The  results  of  even  the  best  of  the  color  re- 
actions must  therefore  be  interpreted  with  great  caution  and  must 
usually  be  regarded  as  of  much  less  significance  than  the  quantita- 
tive numbers. 

On  the  other  hand  a  good  grade  of  arachis  oil,  or  a  carefully 
prepared  mixture  of  lard  oil  with  one  of  the  seed  oils,  can  be  added 
to  olive  oil  in  large  proportion  without  affecting  the  ordinary  con- 
stants to  such  an  extent  as  to  pass  the  limits  which  can  fairly  be 
regarded  as  normal.  It  is  necessary,  therefore,  not  only  to  com- 
pare each  number  found  with  the  established  limits  for  pure  oil, 
but  also  to  view  the  quantitative  results  in  their  relations  to  each 
other  and  to  the  indications  of  the  qualitative  tests. 

While  the  system  to  be  followed  and  the  number  of  tests  re- 
quired will  naturally  vary  in  different  laboratories  the  following 
may  be  recommended.  Determine  accurately  the  specific  gravity, 
the  iodine  number,  and  saponification  number.  If  an  abundance 
of  the  sample  is  at  hand  determine  the  specific  temperature  reac- 
tion and  the  acidity.  The  index  of  refraction  may  be  found,  if 

148 


FATTY    OILS— SPECIAL   METHODS. 


149 


convenient,  but  is  usually  of  less  importance  than  the  other  deter- 
minations. Apply  the  nitric  acid  test,  Halphen's  test,  one  or  more 
of  the  color  reactions  for  sesame  oil,  and  examine  for  arachidic  acid 
by  Renard's  method.  Finally,  if  the  importance  of  the  sample 
justifies  the  time  required,  separate  and  examine  the  unsaponifiable 
matter  and  the  mixed  fatty  acids  and  determine  the  viscosity  of 
the  soap  solution  by  Abraham's  method  described  below. 

ANALYTICAL  PROPERTIES  OF  OLIVE  OIL. 

The  numbers  included  in  the  table  of  "  constants  "  already  given 
are  intended  to  cover  the  range  of  normal  variations  in  oils  found 
in  the  American  markets.  Miintz,  Durand  and  Milliau  *  examined 
samples  from  Africa,  Spain,  Portugal,  Greece,  Turkey,  and  the 
Levant  without  finding  any  significant  variation  in  the  specific 
gravity,  iodine  number,  or  temperature  reaction.  According  to 
Milliau,  Bertainchand  and  Malet,  |  however,  Tunis  oils  have  high 
specific  gravities,  the  average  of  49  samples  being  0.9183.  Tol- 
man  and  Munson  have  recently  examined  a  large  number  of  olive 
oils  many  of  which  were  of  known  origin.  The  following  are  taken 
from  their  results. 


Description. 

Iodine 
Number. 

jjft 

Index  of 
Refraction 
15-5°. 

t| 

||j 

H 

California  oils  of  known  origin      f  A/r?X' 
(42  samples).                                        j  «»; 

89.8 
78.5 
85-3 

0.9180 
0.9162 
0.9170 

.4718 
.4703 
.4713 

109.7 
94-5 

101.8 

8.21* 
O.2O 
1.  2O 

Italian   oils   of  known   origin     J  *,** 
(17  samples). 

86.1 
79.2 
81.6 

0.9180 

0.9155 
0.9163 

.4713 

•4705 

104.7 
95-6 
99.1 

2.79 

0-57 
I.  II 

Italian  oils  (commercial)  not      f  M*X' 
found  adulterated  (57  samples).!    1  AV 

84.5 
77-5 
80.9 

0.9179 
0.9150 
0.9161 

.4712 
.4701 
.4706 

108.4 
88.4 
97-8 

5-30 
0.72 
2.42 

French  oils  (commercial)   not     fjj"' 
found  adulterated  (60  samples).       j    .  ln' 

85.0 
79.0 
81.3 

0.9183* 
0.9150 
0.9166 

.4713 
.4699 
.4708 

114.4 

90.4 

100.  1 

3.63 

o.45 
1-59 

*  Two  samples  with  larger  amounts  of  free  acid  were  found  but  were  excluded  from 
average  as  being  unfit  for  use  as  salad  oils. 

f  Omitting  one  sample  containing  15.25  per  cent,  of  free  acid  and  having  a  specific 
gravity  of  0.9134. 

J  Omitting  one  sample  having  an  abnormally  high  specific  gravity  (0.9196)  which 
may  have  been  due  to  exposure. 

*  Bulletin  du  Ministe're  de  1' Agriculture,  1895.     Quoted  from  Bui.  77,  Bur.  Chem., 
U.  S.  Dept.  Agriculture. 

f  Bulletin  de  1' Agriculture  et  Commerce  de  Tunis.     Quoted  from  Bui.  77,  loc.  cit. 
%  Bui.  77,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


150 


ORGANIC  ANALYSIS. 


The  average  iodine  number  of  the  oils  from  California  is  there- 
fore higher  than  that  of  the  French  or  Italian  oils  and,  as  might  be 
expected,  the  higher  iodine  number  is  accompanied  by  a  higher 
specific  gravity,  refractive  index,  and  temperature  reaction.  Al- 
though individual  samples  will  show  slight  variations,  the  same 
general  relation  is  found  on  comparing  oils  which  differ  in  iodine 
numbers  though  obtained  from  the  same  locality.  Thus  taking  at 
random,  from  among  the  California  oils  of  known  origin  examined 
by  Tolman  and  Munson,  10  samples  with  high  and  10  with  low 
iodine  numbers  the  following  average  figures  were  found : 


Iodine  Number. 

Specific  Gravity 
155° 
'5-5°' 

Index  of  Re- 
fraction 
15-5°. 

Specific    Tem- 
perature 
Reaction. 

First  group. 
Second  group. 

88.44       , 
83.12 

0.9172 
0.9166 

I.47I6 
I.47II 

105-7 
98.6 

The  normal  relations  of  the  constants  to  each  other  and  the 
changes  which  may  occur  as  the  result  of  age  or  exposure  *  must 
always  be  taken  into  consideration  when  interpreting  the  results  of 
an  analysis. 

DETECTION  OF  COTTONSEED  OIL. 

The  presence  of  cottonseed  oil  in  olive  oil  raises  the  specific 
gravity,  iodine  number,  and  temperature  reaction  and  lowers  the 
viscosity  of  the  soap  solution.  A  mixture  of  cottonseed  and  lard 
oils  may,  however,  be  added  to  olive  oil  in  large  quantity  without 
greatly  affecting  any  but  the  last  of  these  "  constants."  The  quali- 
tative tests  for  cottonseed  oil  are  therefore  of  considerable  impor- 
tance. 

Halpheris  Reaction.^ 

Dissolve  I  part  of  sulphur  in  100  parts  of  carbon  bisulphide 
and  mix  the  solution  with  an  equal  volume  of  amyl  alcohol. 

Mix  equal  volumes,  2  to  3  c.c.  each,  of  the  reagent  and  the  oil  to 
be  tested  and  heat  the  test  tube  containing  the  mixture  gently  at 
first  until  violent  boiling  ceases,  then  in  a  bath  of  boiling  saturated 
solution  of  common  salt.  Heat  for  2  hours  unless  a  color  develops 
sooner.  If  cottonseed  oil  is  present  the  solution  turns  orange  or 
red. 

*  See  section  on  this  subject  beyond. 

f  Halphen  :  Ann.  Chim.  Analyt.,  1898,  3,  9;  Analyst,  1898,  23,  131  ;  Bui.  65. 
Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


FATTY    OILS— SPECIAL   METHODS.  151 

This  is  probably  the  most  sensitive  and  characteristic  test  for 
cottonseed  oil  and  the  least  liable  to  give  unsatisfactory  results  in 
the  hands  of  an  inexperienced  person.  The  presence  of  I  or  2  per 
cent,  of  unchanged  cottonseed  oil  in  olive  oil  is  detected  without 
difficulty.  A  reaction  is  often  obtained  *  with  lard  or  lard  oil  or 
even  with  butter  fatf  from  animals  which  have  been  fed  upon  cot- 
tonseed meal.  Copac  (or  kapok)  oil,  which  is  closely  related  to 
cottonseed  oil,  gives  the  same  reaction.  When  heated  at  250°  for 

10  to  20  minutes,  cottonseed  oil  loses  the  property  of  giving  this 
reaction.     No  pure  olive  oil  has  yet  been  known  to  give  a  similar 
coloration.     Hence  a  positive  result  is  considered  conclusive  but 
a  negative  result  is  not.     The  substance  to  which  the  reaction  is 
due  cannot  be  removed  by  treatment  with  animal  charcoal  J  and  is 
supposed  to  be  an  unsaturated  acid  which  combines  with  sulphur 
giving  a  red  compound.  § 

Nitric  Acid  Test. 

Cottonseed  oil  shaken  at  room  temperature  with  an  equal  vol- 
ume of  nitric  acid,  of  1.37  to  1.38  specific  gravity,  gives  a  brown 
coloration,  sometimes  only  on  standing  over  night.  Other  seed 
oils  give  similar  reactions.  Normal  olive  oil  under  the  same 
treatment  shows  no  change  of  color. 

The  test  is  not  so  delicate  as  that  of  Halphen  but  is  applicable  to 
cottonseed  oil  which  has  been  heated  until  it  no  longer  colors  the 
Halphen  reagent.  It  may  also  be  of  value  in  determining 
whether  a  weak  test  with  the  latter  reagent  is  due  to  a  small 
amount  of  unheated  cottonseed  oil  or  to  a  larger  amount  which 
has  been  heated  sufficiently  to  weaken  the  Halphen  reaction 
(Tolman  and  Munson).  According  to  Lewkowitsch  this  reaction 
cannot  be  relied  upon  to  detect  less  than  10  to  20  per  cent,  of  Ameri- 
can cottonseed  oil  in  olive  oil.  Tolman  and  Munson  consider  the 
test  much  more  delicate.  A  positive  result  should  always  be  con- 
firmed by  finding  a  high  iodine  number  or  by  proving  the  presence 
of  some  other  oil  having  a  very  low  iodine  number ;  for  pure  olive 

011  if  much  altered  as  the  result  of  age  and  exposure  will  some 

*Soltsein:  Ztschr.  offentl.  Chem.,  1901,  7,  140. 

f  Wauters  :  Bull.  Assoc.  Belg.  Ghent. ,  13,  404;  Joitrn.  Soc.  Chem.  Ind.y  1900 
19,  172. 

{  Utz  :   Chem.  Rev.  Fett.  und Harz.  Ind.,  1902,  9,  125  ;    Gill's  Oil  Analysis,  p.  73. 

§  Raikow  :  C/iim.  Z(g.,  1 900,  24,  562,  583;  192,  26,  10.  See  also  Halphen: 
Bull.  Soc.  Chem.,  1905,  [3],  33,  108. 


152  ORGANIC  ANALYSIS. 

times  give  a  reaction  which  cannot  be  distinguished  from  that  of 
cottonseed  oil. 

DETECTION  OF  ARACHIS  (PEANUT)  OIL. 

Arachis  oil  has  usually  a  higher  specific  gravity  and  iodine  num- 
ber and  practically  always  a  higher  temperature  reaction  than  olive 
oil.  The  specific  temperature  reaction  of  arachis  oil  with  concen- 
trated sulphuric  acid  is  usually  40  to  60  units  higher  than  the 
iodine  number  of  the  same  sample,  whereas  olive  oil  usually  shows 
a  difference  of  less  than  20  and  very  rarely  of  more  than  25  units 
between  the  iodine  and  the  specific  Maumene  numbers.  The  pres- 
ence of  peanut  oil  in  olive  oil  greatly  diminishes  the  viscosity  of  the 
soap  solution  obtained  on  saponification.  Any  of  these  changes, 
however,  might  be  due  to  other  adulterants.  Arachis  oil  is  shown 
conclusively  by  isolating  and  identifying  arachidic  acid.  This  can 
be  done  with  approximately  quantitative  results  by  Tolman's 
modification  of  Renard's  method. 

Reward-  Tolman  lest  for  Arachidic  Acid.  * 

Weigh  20  grams  of  oil  in  an  Erlenmeyer  flask.  Saponify  with 
alcoholic  potash,  neutralize  exactly  with  dilute  acetic  acid, 
using  phenolphthalein  as  indicator,  and  wash  the  solution  in  a 
500  c.c.  flask  containing  a  boiling  mixture  of  100  c.c.  of  water  and 
1 20  c.c.  of  a  20  per  cent,  lead  acetate  solution.  Boil  one  minute 
and  then  cool  by  immersing  the  flask  in  water,  occasionally  giving 
it  a  whirling  motion  to  cause  the  precipitated  lead  soaps  to  stick 
to  the  sides  of  the  flask.  After  thorough  cooling,  pour  off  the 
water  containing  the  excess  of  lead  acetate  and  wash  the  soap  with 
cold  water  and  then  with  90  per  cent,  alcohol.  After  pouring  off 
the  alcohol  as  completely  as  possible,  add  200  c.c.  of  ether,  cork 
the  flask  and  allow  to  stand  until  the  soap  is  disintegrated,  then 
connect  with  a  reflux  condenser,  heat  gently  to  boiling  and  boil  for 
5  minutes  on  a  safety  water-bath  or  an  electric  heater.  Cool  to 
15°  and  allow  to  stand  over  night. 

Filter,  wash  the  residue  thoroughly  with  ether,  and  then  trans- 
fer it  from  the  filter  to  the  flask  by  means  of  a  stream  of  hot  water 
acidified  with  hydrochloric  acid.  Add  an  excess  of  dilute  hydro- 
chloric acid  and  200  c.c.  of  hot  water  and  heat  until  the  fatty  acids 

*  Renard  :   Compt.  rend.,   1871,  73,  1330. 

Tolman:  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  64.  See  also  Arch- 
butt:  Journ.  Soc.  Chem.  Ind.,  1898,  17,  1124. 


FATTY    OILS— SPECIAL   METHODS.  153 

separate  as  a  clear  oily  layer.  Nearly  fill  the  flask  with  hot  water 
and  allow  to  stand  at  room  temperature  until  the  layer  of  fatty 
acids  has  completely  separated  and  solidified.  Remove  and  drain 
the  cake  of  fatty  acids,  wash  again  with  hot  water,  then  dissolve 
in  100  c.c.  of  boiling  alcohol,  90  per  cent,  by  volume.  Cool  the 
solution  to  15°,  shaking  frequently,  and  allow  it  to  stand  as  long 
as  any  acid  continues  to  crystallize  out,  or  over  night,  at  a  temper- 
ature not  above  20°.  Filter,  wash  the  crystals  twice  with  10  c.c. 
of  90  per  cent,  alcohol,  noting  the  total  volume  of  filtrate  and 
washings,  and  then  with  alcohol,  70  per  cent,  by  volume  (in  which 
the  crystals  are  practically  insoluble).  Dissolve  the  crystals  by 
means  of  hot  absolute  alcohol  in  a  weighed  dish,  evaporate,  dry, 
and  weigh.  To  this  weight  add  0.0045  gram  for  each  10  c.c.  of 
90  per  cent,  alcohol  in  the  filtrate  and  washings  if  the  temperature 
of  filtration  was  20°;  or  0.0025  gram  for  each  10  c.c.  if  the  temper- 
ature was  15°. 

The  melting  point  of  arachidic  acid  obtained  in  this  way  is  71° 
to  73°.  According  to  Tolman  and  Munson  *  the  determination  of 
the  melting  point  must  not  be  neglected  since  cottonseed  and  lard 
oils  have  been  found  to  give  crystals  resembling  arachidic  acid  in 
appearance,  but  having  a  lower  melting  point.  Tolman  finds  that 
from  5  to  10  per  cent,  of  the  oil  can  be  detected  by  this  method. 
On  the  usual  assumption  that  the  oil  yields  5  per  cent,  of  the  acid,f 
each  centigram  found  as  described  above  (using  20  grams  of  oil) 
indicates  I  per  cent,  of  arachis  (peanut)  oil  in  the  sample. 

DETECTION  OF  SESAME  OIL. 

Sesame  oil  affects  the  usually  determined  constants  in  the  same 
way  as  cottonseed  oil  and  to  practically  the  same  extent.  The 
color  reactions  are  usually  considered  quite  characteristic. 

Boudouiffs  Test. 

Dissolve  O.I  gram  of  sugar  in  10  c.c.  of  hydrochloric  acid  of 
1. 1 8  to  1. 20  specific  gravity  and  add  20  c.c.  of  the  oil.  Shake 
thoroughly  in  a  test  tube  for  one  minute  and  allow  to  stand.  The 
water  solution  separates  quickly  and  shows  a  distinct  red  or 
crimson  color  if  the  sample  contains  I  per  cent,  or  more  of  sesame 

*U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  77,  p.  35. 

I  This  is  probably  more  nearly  a  maximum  than  an  average  yield.  Tolman  and 
Munson  (loc.  cit.}  obtained  from  3.41  to  4.24  per  cent. 


154  ORGANIC  ANALYSIS. 

oil.     The  active  reagent  is  probably  furfurol  formed  by  the  action 
of  the  acid  upon  the  sugar. 

Villivecchict  s  modification  consists  in  shaking  10  c.c.  of  the  oil 
with  10  c.c.  of  acid  (1.20  sp.  gr.)  to  which  has  been  added  O.I  c.c. 
of  a  2  per  cent,  solution  of  furfurol  in  95  per  cent,  alcohol. 

Olive  oils  of  known  purity  have  usually  been  found  to  give  only 
a  slight  pink  color,  but  sometimes  the  reddening  of  the  water 
solution  is  so  pronounced  as  to  cause  confusion  with  that  produced 
by  a  small  amount  of  sesame  oil.  Check  experiments  therefore 
should  always  be  made.  If  much  sesame  oil  is  present  the  red 
color  should  be  perceptible  in  the  oily  layer  as  well  as  in  the  water 
solution. 

Tocher  s  lest. 

Dissolve  I  gram  of  pyrogallol  in  15  c.c.  of  concentrated  hydro- 
chloric acid.  Shake  this  solution  with  15  c.c.  of  oil  in  a  separatory 
funnel  and  allow  to  stand  for  I  or  2  minutes.  Draw  off  the  aqueous 
solution  and  boil  for  5  minutes.  The  presence  of  sesame  oil  is 
indicated  if  the  solution  after  boiling  appears  red  by  transmitted 
and  blue  by  reflected  light. 

This  test  has  not  been  so  generally  used  nor  so  thoroughly 
studied  as  the  preceding.  The  Association  of  Official  Agricul- 
tural Chemists  authorize  the  use  of  either  Boudouin's,  Villivec- 
chia's,  or  Tocher's  test  for  the  detection  of  sesame  oil  in  edible 
oils  and  fats. 

DETECTION  OF  RAPESEED  OIL. 

The  acrid  taste  of  this  oil  would  prevent  the  use  of  any  but 
highly  refined  grades  in  salad  oils  and  since,  in  America  at  least, 
refined  rape  oil  is  little  if  any  cheaper  than  olive,  it  is  not  likely  to 
be  largely  used  as  an  adulterant.  The  presence  in  the  oil  of  rape, 
and  other  seeds  of  the  Cruciferce>  of  large  proportions  of  an  acid 
(erucic)  having  a  much  higher  molecular  weight  than  oleic  and 
linoleic,  results  in  a  low  saponification  figure  and  a  high  index  of 
refraction  together  with  low  specific  gravity.  The  determination 
of  these  constants  would  probably  show  rape  oil  in  olive  oil  if  20 
per  cent,  or  more  were  present. 

Rapeseed  oil  is  less  soluble  than  olive  oil  in  acetic  acid,  but 
mixtures  containing  20  to  40  per  cent,  of  rape  oil  cannot  be  dis- 
tinguished from  olive  oil  by  this  test  (Tolman). 

Color  reactions  for  rapeseed  oil  have  been  proposed  but  have 
not  given  satisfactory  results. 


FATTY    OILS— SPECIAL   METHODS.  155 

DETECTION  OF  MAIZE,  POPPYSEED,  AND  LARD  OILS. 

Each  of  these  oils  has  a  characteristic  odor  or  taste,  the  odor  of 
lard  oil  being  intensified  by  heating.  These  properties,  however, 
cannot  be  relied  upon  as  the  substances  to  which  they  are  due  can 
be  almost  entirely  eliminated  in  the  refining  process.  The  effect 
of  maize  or  poppy  oil  in  raising  the  iodine  number  would  be  very 
noticeable  but  might  be  neutralized  by  the  addition  of  a  somewhat 
greater  quantity  of  lard  oil.  The  difference  between  the  iodine 
number  and  the  specific  temperature  reaction  would  be  appreciably 
greater  in  such  a  mixture  than  in  pure  olive  oil  (compare  detection 
of  arachis  oil).  Maize  and  poppyseed  oils  react  with  nitric  acid  giv- 
ing brown  colors  similar  to  that  produced  by  cottonseed  oil.  Lard 
oil  often  gives  the  same  reaction  and  might  be  indicated  by  the 
melting  point  of  the  fatty  acids  (those  of  lard  oil  having  a  rela- 
tively high  melting  point,  33°  to  38°  according  to  Tolman  and 
Munson)  or  by  the  character  of  the  unsaponifiable  matter  —  phy- 
tosteryl  acetate  test.* 

All  of  these  oils  (as  well  as  most  others)  yield  soap  solutions  of 
much  lower  viscosity  than  those  obtained  from  pure  olive  oils.| 
The  determination  and  significance  of  this  property  has  recently 
been  studied  in  some  detail  J  and  the  method  has  been  found 
capable  of  giving  valuable  results,  especially  if  the  conditions 
worked  out  by  Abraham  are  carefully  observed.  For  a  full  dis- 
cussion of  these  conditions  the  original  paper  must  be  consulted. 
The  essential  features  of  the  process  are  as  follows  : 

Abrahams  Modification  of  Blasdales  Viscosity  Test. 

The  saponification  number  having  been  determined,  weigh  3 
grams  of  oil  in  an  accurately  graduated  100  c.c.  flask,  add  2  c.c. 
of  alcohol  and  an  amount  of  standard  potash  solution  sufficient  to 
saponify  the  oil  and  leave  an  excess  of  0.625  gram  of  potassium 
hydroxide.  Close  the  flask  with  a  stopper  having  a  Kroonig  valve 
and  saponify  on  a  water-bath.  After  saponification  expel  the  alco- 
hol by  warming  and  allowing  air  freed  from  carbon  dioxide  to  pass 
through  the  flask,  while  a  partial  vacuum  is  maintained  by  means 
of  a  suction  pump.  In  this  way  the  alcohol  is  entirely  removed 
in  5  to  10  minutes.  Evaporation  should  not  be  carried  to  com- 

*B6mer:  Ztschr.  Unters.  Nahr.-Genussm.,  1901,  4,  1091. 

f  Blasdale  :  Journ.  Amer.  Chevi.  Soc.,  1895,  J7>  937- 

J  Abraham  :  Ibid.,  1903,  25,  968.     Sherman  and  Abraham  :  Ibid.,  1903,  25,  977. 


156  ORGANIC  ANALYSIS. 

plete  dryness.  Without  allowing  the  flask  to  cool,  add  50  c.c.  of 
hot  water,  rotate  gently  until  a  homogeneous  solution  of  the  soap 
is  obtained,  cool  to  20°,  fill  to  the  mark  with  distilled  water  and 
mix  well  by  shaking  or  by  repeatedly  pouring  the  solution  from 
one  flask  to  another.  Determine  the  viscosity  of  the  solution  in  an 
Ostwald  viscosimeter  immersed  in  water  kept  at  20°.  Care  must 
be  taken  to  avoid  the  introduction  of  air  bubbles  into  the  viscosim- 
eter and  to  maintain  the  exact  temperature.  Repeat  the  readings 
until  five  or  more  concordant  results  are  obtained.  The  viscosim- 
eter is  standardized  by  means  of  distilled  water  and  it  is  advis- 
able to  select  for  this  work  an  instrument  in  which  the  time  of 
flow  of  water  is  about  100  seconds.  Successive  readings  of  a 
soap  solution  should  then  agree  within  2  seconds. 
The  viscosity  is  calculated  as  follows  : 


where 

vl  =  the  viscosity  number. 
tl  =  the  time  of  flow  (in  seconds). 
jj  =  specific  gravity  of  the  solution. 
t  =  time  of  flow  of  distilled  water. 

The  viscosity  numbers  obtained  by  this  method  were  : 

Olive  oil  of  known  purity  (9  samples)  ..........................  168.0-185.7 

Olive  oil  of  doubtful  purity  (4  samples)  ........................  145.8-165.8 

Lard  oil  (5  samples)  .....................  ...........................  122.9-135.0 

Arachis  (peanut)  oil  (i  sample)  .....................  .  ...........  126.6 

Cottonseed  oil  (3  samples)  .......................................  126.6-127.9 

Rapeseed  oil             "           ........................................  124.7-125.7 

Sesame  oil  (i  sample)         .........................................  I39-2 

Maize  oil             "                  ........................................  126.6 

Poppyseed  oil      "                 ........................................  I23-9 

Mixtures  of  olive  and  lard  oils  gave  viscosity  numbers  agreeing 
closely  with  those  obtained  by  calculation,  but  cottonseed  or 
arachis  oil  when  added  to  olive  oil  lowered  the  viscosity  to  a  much 
greater  extent  than  would  have  been  predicted,  indicating  that  the 
viscosity  number  is  a  more  useful  means  of  detecting  adulteration 
than  appears  from  a  comparison  of  the  results  obtained  on  testing 
the  olive  oil  and  its  adulterants  separately. 

The  usefulness  of  the  method  for  testing  isolated  samples  is 
limited  by  the  fact  that  comparable  results  can  be  obtained  only 


FATTY    OILS— SPECIAL   METHODS.  i  $7 

under  strictly  uniform  conditions,  the  viscosity  of  the  soap  solu- 
tion being  greatly  influenced  by  slight  variations  in  strength, 
alkalinity,  or  temperature,  while  the  terms  in  which  the  results  are 
expressed  will  naturally  vary  with  the  form  of  viscosimeter  used ; 
but  in  cases  of  sufficient  importance  to  justify  the  time  required 
to  arrange  the  apparatus  and  make  comparative  determinations, 
the  "  viscosity  number  "  will  be  found  an  important  factor  in  the 
examination  of  olive  oil  for  adulterants. 

DRYING    OILS. 

While  other  oils,  including  especially  poppyseed  and  tung  oils, 
are  used  as  drying  oils  for  special  purposes,  linseed  oil  is  of  much 
greater  importance,  being  preferred  in  the  great  majority  of  cases 
in  which  a  drying  oil  is  needed.  Only  linseed  oil  and  its  adulter- 
ants will  be  considered  here. 

ANALYTICAL  PROPERTIES  OF  LINSEED  OIL. 

Commercial  linseed  oil  is  usually  designated  by  the  region  of  its 
origin.  It  varies  considerably,  the  variation  being  due  mainly  to 
the  presence  of  foreign  seeds  in  the  linseed  at  the  time  of  pressing. 
Hempseed  is  practically  always  present,  sometimes  in  very  small 
proportion  but  often  to  the  extent  of  5  per  cent,  or  more,  of  the 
weight  of  seed.  The  drying  properties  of  linseed  oil  are  better  the 
purer  the  seeds  from  which  it  is  pressed.  The  iodine  number  and 
the  drying  power  of  the  oil  decrease  as  the  proportion  of  hempseed 
increases.  Hence  a  linseed  oil  containing  much  hemp  oil 
would  be  shown  to  be  of  inferior  quality  by  its  low  iodine  num- 
ber but  could  not  be  pronounced  adulterated  so  long  as  this  did 
not  fall  below  170.  The  maximum  iodine  number  of  linseed  oil  is 
difficult  to  fix  since  the  results  obtained  by  the  method  of  Wijs 
often  exceed  the  Hubl  numbers,  but  since  no  other  common  oil 
has  a  higher  iodine  number  than  linseed,  the  maximum  limit  is  of 
little  practical  importance  in  the  detection  of  adulterations. 

The  usual  range  of  the  more  important  analytical  constants  of 
linseed  oil  has  been  given  in  the  table  at  the  end  of  the  preceding 
chapter.  The  interpretation  of"  constants,"  their  relations  to  each 
other,  and  their  use  in  the  detection  of  adulterations  having  been 
discussed  in  some  detail  in  connection  with  the  examination  of 
salad  oils,  it  will  be  sufficient  in  this  case  to  mention  briefly  the 
principal  adulterants  with  means  for  the  detection  of  each,  and  de- 
scribe the  hexabromide  test  which  distinguishes  linseed  from 
practically  all  other  oils. 


158  ORGANIC  ANALYSIS. 

ADULTERANTS  AND  METHODS  OF  DETECTION. 

Mineral  Oil. 

Mineral  oil  would  greatly  lower  the  iodine  number,  temperature 
reaction,  and  saponification  number.  Whenever  a  low  saponifica- 
tion  number  is  found  the  unsapomfiable  matter  should  be  sepa- 
rated and  examined.  Any  mineral  oil  which  is  not  volatile  &t 
100°  can  be  separated  quantitatively,  dried,  and  weighed.  Volatile 
mineral  oil  can  be  distilled  by  means  of  a  current  of  steam,  sepa- 
rated from  water  in  the  distillate,  and  measured  or  weighed.  In 
case  turpentine  were  present,  as  in  some  so-called  "  boiled  "  oils,  it 
would  be  distilled  with  steam  and  would  separate  from  water  in  the 
distillate  in  the  same  way.  The  optical  rotatory  power  of  turpen- 
tine affords  an  easy  means  of  distinguishing  it  from  benzine  or 
other  volatile  mineral  oil. 

Rosin  and  Rosin  Oil. 

Rosin  dissolved  in  linseed  oil  raises  the  specific  gravity  and  index 
of  refraction  while  the  saponification  and  iodine  numbers  are  ap- 
preciably decreased  only  when  large  amounts  of  rosin  are  added. 
Presence  of  rosin  greatly  increases  the  acid  number  which  in  pure 
linseed  oil  is  usually  less  than  7.  .Rosin  acids  can  be  separated 
and  determined  by  Twitchell's  method  as  described  under  soap 
analysis  beyond. 

Rosin  oil  in  linseed  oil  would  raise  the  specific  gravity  and 
greatly  lower  the  saponification  and  iodine  numbers.  Rosin  oil  is 
a  mixture  of  substances  many  of  which  are  unsaponifiable,  so  that 
its  presence  in  linseed  oil  would  increase  the  amount  of  unsaponi- 
fiable matter. 

Either  rosin  or  rosin  oil  can  be  detected  by  the  Liebermann- 
Storch  color  reaction  or  by  determining  the  bromine  substitution 
number. 

Liebermann-Storch  Reaction. — Shake  2  c.c.  of  the  oil  with  5  c.c. 
of  acetic  anhydride,  warming  gently.  Allow  to  cool,  draw  off  the 
anhydride  and  test  by  adding  one  drop  of  sulphuric  acid  (i :  i).  A 
violet  color  (not  permanent)  is  produced  in  the  presence  of  rosin 
or  rosin  oil.  Cholesterol,  which  might  be  found  in  linseed  oil  if 
fish  oil  were  present  as  an  adulterant,  gives  a  similar  color  reaction. 

Bromine  Substitution  Number  (Mcllhiney).  —  Fatty  oils  take  up 
bromine  by  direct  addition,  little  or  no  substitution  taking  place. 
With  rosin  and  rosin  oil  much  the  greater  part  of  the  bromine  is 


FATTY    OILS— SPECIAL   METHODS.  159 

taken  up  by  substitution,  a  molecule  of  hydrobromic  acid  being 
formed  for  each  molecule  of  bromine  which  disappears.  The 
hydrobromic  acid  thus  affords  a  means  of  measuring  the  amount 
of  substitution.  It  is  determined  by  adding  an  excess  of  potassium 
iodate  and  titrating  the  liberated  iodine.  The  same  apparatus  can  be 
used  as  in  the  determination  of  the  iodine  number  and  the  manipula- 
tion is  similar.  From  0.2  to  0.3  gram  of  the  drying  oil  is  dissolved 
in  10  c.c.  of  carbon  tetrachloride  and  20  c.c.  of  a  one-third  normal 
solution  of  bromine  in  carbon  tetrachloride  is  added.  After  two 
minutes  potassium  iodide  is  added  and  the  excess  of  halogen 
titrated  by  means  of  thiosulphate  as  in  the  determination  of  the 
iodine  number.  This  shows  the  total  amount  of  bromine  which 
has  disappeared.  As  soon  as  this  titration  is  finished,  add  5  c.c. 
of  a  2  per  cent,  solution  of  potassium  iodate  and  titrate  the  iodine 
set  free  from  the  iodate  by  the  action  of  the  free  halogen  acids, 
according  to  the  reaction  :  6HI  +  KIO3  =  3!,  -f  KI  +  3H2O. 

The  bromine  thus  found  is  equal  in  amount  to  that  which  has 
combined  with  the  sample  by  substitution.  For  further  details  of 
manipulation  the  reader  must  be  referred  to  the  original  papers.* 
According  to  Mcllhiney  the  bromine  substitution  number  of  raw 
or  boiled  linseed  oil  is  always  less  than  7  while  rosin  oil  gives 
numbers  from  40  to  100  and  rosin  from  65  to  80. 

Maize  Oil. 

The  presence  of  maize  oil  in  linseed  oil  lowers  the  specific 
gravity,  index  of  refraction,  iodine  number,  and  temperature  reac- 
tion. The  amount  which  can  be  added  without  carrying  these 
numbers  below  the  normal  limits  of  variation  will  depend  upon  the 
quality  of  the  linseed  oil  in  the  mixture.  Since  the  maize  oil  used 
as  an  adulterant  of  linseed  would  probably  not  be  highly  refined, 
the  characteristic  odor  and  taste  would  aid  in  its  detection. 

Cottonseed  Oil. 

The  "  constants  "  of  linseed  oil  would  be  lowered  by  cottonseed 
in  the  same  way  as  by  maize  oil  and  to  a  somewhat  greater  extent. 
If  the  cottonseed  oil  had  not  been  heated,  its  presence  would  be 
detected  by  the  Halphen  test  as  described  under  salad  oils. 

*  Mcllhiney  :  Journ.  Amer.  Chem.  Soc.,  1894,  16,  275;  1899,  21,  1084;  1902, 
24,  1109.  See  also  Tolman  :  Ibid.,  1904,  26,  826. 


i6o  ORGANIC  ANALYSIS. 

Fish  Oils. 

Menhaden  and  other  fish  oils  are  often  used  as  adulterants  and  are 
difficult  to  detect  with  certainty  since  their  "  constants  "  are  fre- 
quently within  the  limits  found  for  pure  linseed  oil.  Their  presence 
is  often  indicated  by  the  odor,  but  this  cannot  be  relied  upon  as 
the  difference  in  odor  between  refined  menhaden  and  low  grade 
linseed  oil  is  not  so  pronounced  as  might  be  supposed.  Lewko- 
witsch  recommends  the  determination  of  the  melting  point  of  the 
phytosteryl  acetate  obtained  from  the  oil,  and  also  that  of  the 
hexabromide  of  the  fatty  acids  (see  below).  The  crystals  of  phy- 
toseryl  acetate  from  pure  linseed  oil  melt  at  128°-!  29°  (Bomer  and 
Winter)  while  in  the  presence  of  cholesterol  from  fish  oil  much 
lower  melting  points  are  obtained. 

HEXABROMIDE  TEST. 

Hehner  and  Mitchell  *  showed  that  linseed  and  fish  oils  differ 
from  other  oils  in  yielding  considerable  quantities  of  insoluble  hexa- 
brom-addition  products  when  treated  with  bromine  in  ether  solu- 
tion. They  applied  the  test  directly  to  the  oil  as  follows : 

Dissolve  I  to  2  grams  of  oil  in  40  c.c.  of  ether  acidulated  with 
glacial  acetic  acid,  cool  the  solution  to  5°  and  add  bromine,  drop 
by  drop,  until  the  solution  is  permanently  colored  brown.  After 
standing  for  3  hours,  filter  on  asbestos  and  wash  successively  with 
5  c.c.  each  of  cold  glacial  acetic  acid,  alcohol,  and  ether.  Dry  the 
precipitate  at  100°  and  weigh. 

Linseed  oil  yields  23  to  38  per  cent,  of  hexabromide,  the  amount 
increasing  with  the  iodine  number  of  the  oil.  Some  of  the  fish 
oils  yield  equal  or  greater  amounts,  but  tung,  poppy,  and  walnut 
oils,  and  such  seed  oils  as  maize  and  cottonseed,  yield  almost  no 
hexabromide,  according  to  the  figures  compiled  by  Lewkowitsch 
never  over  2  per  cent. 

Lewkowitsch  recommends  t  that  the  test  be  applied  to  the  mixed 
fatty  acids  rather  than  to  the  oil  itself.  In  the  separation  of  the 
acids  care  must  be  taken  to  avoid  oxidation  by  exposure  to  air. 
The  mixture  of  fatty  acids  from  linseed  oil  yields  30  to  42  per  cent, 
of  hexabromide  melting  to  a  clear  liquid  at  1/5°  to  180°,  whereas 
the  corresponding  products  from  fish,  liver,  and  blubber  oils  do  not 
melt  at  this  temperature  but  become  darker  and  are  completely 

*  Analyst,  1898,  23,310. 

fOils,  Fats,  and  Waxes  (3d  Ed.),  P-  459. 


FATTY    OILS— SPECIAL   METHODS. 


161 


blackened  at  about  200°.     Lewkowitsch  states  that  this  test   is 
capable  of  showing  the  presence  of  10  per  cent,  of  fish  oil  in  linseed. 

OILS   ALTERED    BY   AGE   OR   OXIDATION. 

It  has  been  assumed  in  discussing  the  analytical  "  constants  " 
that  the  oils  under  examination  are  fresh  or  have  been  kept  under 
such  conditions  as  to  prevent  any  material  alteration.  Age  alone 
probably  has  no  appreciable  effect  upon  the  analytical  properties 
of  commercially  pure  fatty  oils,  but  such  oils  when  kept  for  a  long 
time  in  contact  with  air,  for  example,  in  partially  filled  or  loosely 
stoppered  vessels,  take  up  atmospheric  oxygen  and  gradually  be- 
come considerably  altered  in  those  properties  which  are  commonly 
regarded  as  "  constants."  This  atmospheric  oxidation  naturally 
takes  place  much  more  rapidly  with  drying  than  with  non-drying 
or  semi-drying  oils,  and  in  open  vessels  than  in  those  in  which  the 
oil  is  exposed  to  only  a  limited  amount  of  air.  It  is  probable  that 
oils  which  have  been  thus  altered  are  of  much  more  frequent 
occurrence  in  commerce  than  has  been  supposed. 

The  influence  of  such  oxidation  upon  the  more  important  ana- 
lytical properties  is  to  increase  the  specific  gravity,  index  of  re- 
fraction, and  temperature  reaction  with  sulphuric  acid,  and  to  de- 
crease the  iodine  number,  the  specific  refractive  power  *  and,  in  the 
case  of  olive  oil,  the  viscosity  of  the  soap  solution.  The  acidity  of 
the  oil  may  increase  at  the  same  time  but  this  change  does  not 
always  occur. 

The  following  results  were  obtained  upon  oils  intentionally  ex- 
posed to  the  air : 


Oil. 

Iodine 

Sp.  Gr. 
iS-5° 

Index  of 
Refraction 

Specific 
Refractive 

Specific 
Tempera- 

15-5° 

at  15.5°. 

Power. 

Reaction. 

Olive  oil  before  exposure. 

83.8 

0.9165 

.4712 

0.5141 

100 

Same  after  exposure. 

77-3 

0.9240 

.4722 

0.5100 

127* 

Lard  oil  before  exposure. 

73-3 

0.917 

.4697 

O.5I22 

1  06 

Same  after  exposure. 

56.2 

0-943 

.4724 

0.5010 

141 

Cottonseed  before  exposure. 

105.2 

0.923 

•4737 

0.5132 

171 

Same  after  exposure. 

90.2 

0-939 

•4779 

0.5090 

217* 

Linseed  before  exposure. 

177.1 

0-934 

.4835 

0.5177 

Same  after  exposure. 

136.9 

0.969 

.4886 

0.5042 

*  These  numbers  were  determined  earlier  than  the  other  results  on  the  exposed 
samples  and  therefore  do  not  show  the  full  effect  of  the  change. 


*  Calculated  by  Landolt's  formula 
deut.  chem.  Ges.,  1882,  15,  1031.) 


N —  I     .          Index  of  refraction  —  I 
D     '  Specific  gravity 


(Ber. 


1 62  ORGANIC  ANALYSIS. 

Many  other  oils  have  been  tested  with  similar  results.  It  is 
evident  that  oils  thus  altered  are  very  likely  to  be  misjudged, 
especially  if  only  one  or  two  quantitative  determinations  are  made. 
Thus  if  only  the  specific  gravity  and  temperature  reaction  of  the 
olive  oil  had  been  determined  the  results  would  have  been  inter- 
preted as  indicating  the  presence  of  some  seed  oil.  The  iodine 
number  of  the  linseed  oil  taken  alone  would  indicate  extensive 
adulteration  with  some  oil  of  lower  drying  power.  The  results 
emphasize  the  importance  of  determining  the  specific  gravity  and 
the  iodine  number  in  all  cases  and  show  the  advantage  of  deter- 
mining the  temperature  reaction  not  as  a  substitute  for  the  iodine 
number  but  for  comparison  with  it.  For  a  fuller  discussion  of  this 
subject  with  the  results  obtained  upon  a  number  of  other  oils  the 
reader  is  referred  to  two  papers  in  the  Journal  of  the  American 
Chemical  Society  (July,  1903,  and  May,  1905). 

As  the  result  of  all  of  this  work  it  appears  that  the  increase  in 
specific  gravity  and  the  decrease  in  iodine  number  are  almost 
exactly  proportional  to  each  other  in  non-drying  and  semi-drying 
oils,  so  that  in  examining  an  altered  oil  belonging  to  either  of 
these  classes  the  original  iodine  number  can  be  estimated  by 
adding  O.8  to  the  number  found  on  the  exposed  sample  for  each 
increase  of  o.ooi  in  the  specific  gravity.  When  the  original  specific 
gravity  is  not  known  the  calculation  must  be  based  upon  the 
average  specific  gravity  for  oil  of  the  species  under  examination. 
The  error  in  this  assumption  can  hardly  be  sufficient  to  affect  the 
interpretation  of  the  results. 

The  iodine  numbers  of  exposed  samples  of  linseed  and  fish  oils 
cannot  be  corrected  accurately  by  the  rule  given  for  semi-drying 
and  non-drying  oils,  the  number  thus  obtained  being  always  too 
low. 

Commercial  "  blown  "  oils  show  greatly  increased  specific  gravi- 
ties and  decreased  iodine  numbers;  the  changes  appear  to  bear 
much  the  same  relation  to  each  other  as  in  the  oils  which  have  been 
altered  by  exposure. 

EXAMINATION   OF   AN   UNKNOWN   OIL. 

In  the  examination  of  an  unknown  oil  the  specific  gravity  and 
the  iodine  and  saponification  numbers  should  be  determined,  and 
the  appearance,  odor,  and  taste  compared  with  those  of  typical 
oils  of  known  purity.  The  results  thus  obtained  usually  suffice  to 


FATTY    OILS— SPECIAL    METHODS.  163 

locate  the  sample  as  one  of  a  small  group  of  oils,  after  which  any 
special  tests  available  for  the  detection  of  individual  members  of 
the  group  can  be  applied.  The  tests  described  in  this  chapter 
taken  in  connection  with  the  quantitative  determinations  men- 
tioned enable  the  analyst,  in  the  majority  of  cases,  to  identify  the 
oil  or,  if  a  mixture,  the  principal  constituent. 

If  the  saponification  number  indicates  that  only  fatty  oil  is  present, 
but  the  relation  of  the  specific  gravity  to  the  iodine  number  does 
not  correspond  to  that  ordinarily  found  in  any  pure  oil,  the  deter- 
mination of  the  specific  temperature  reaction  and  the  acidity  will 
usually  show  whether  the  discrepancy  is  to  be  attributed  to  oxida- 
tion or  adulteration. 

The  relative  commercial  value  will  of  course  determine  what 
oils  can  profitably  be  used  as  adulterants.  Prices  vary  greatly  in 
different  markets,  as  well  as  with  the  degree  to  which  the  oils  are 
refined,  and  are  also  likely  to  fluctuate  from  year  to  year  so  that 
no  fixed  order  of  commercial  value  can  be  given. 

The  orders  of  commercial  value  given  by  Gill  and  by  Lewkowitsch 
show  considerable  variation,  which  doubtless  is  due  largely  to  the 
differences  between  American  and  English  markets.  In  each  of 
the  lists  the  highest  priced  oils  are  given  first. 

Gill.  —  Almond,  castor,  sesame,  neatsfoot,  rape,  olive,  sperm, 
whale,  peanut  (arachis),  linseed,  tallow,  lard,  fish,  cottonseed, 
mineral,  rosin  oil. 

Lewkowitsch.  —  Almond,  sperm,  olive,  neatsfoot,  lard,  cod  liver, 
arctic  sperm,  arachis,  poppy,  sesame,  seal,  rape,  castor,  cottonseed, 
maize,  linseed,  whale,  fish,  mineral,  rosin  oil. 

In  the  examination  of  mixtures  containing  other  than  fatty  oils, 
it  may  be  necessary  to  separate  the  mixed  fatty  acids  and  examine 
this  mixture  in  order  to  identify  the  fatty  oils  originally  present. 
The  "  constants  "  of  the  fixed  fatty  acids  of  various  oils,  as  well  as 
of  many  oils  and  fats  not  mentioned  in  this  work,  are  conveniently 
tabulated  in  Lewkowitsch's  Laboratory  Companion  to  the  Fat  and 
Oil  Industries. 

ADDITIONAL   REFERENCES. 
OLIVE  OIL. 

Tortelli  and  Ruggeri :  Ztsclir.  angew.  Chem.,  1898,  464. 
Cutolo  :  Rev.  Intern.  Falsif.,  1901,    14,  146;  Journ.   Chem.  Soc., 
1902,  82,  ii,  184. 


1 64  ORGANIC  ANALYSIS. 

Tolman:  Journ.  Amer.  Chem.  Soc.,  1902,  24,  396. 
Gill  and  Tufts:  Ibid.,  1903,  25,  498. 
Tolman  and  Munson :  Ibid.,  1903,  25,  954. 
Mastbaum :  Chem.  Rev.  Fett  und  Harz  hid.,  1904,  u,  39,  64 ; 
Ztschr.  Unters.  Nahr.-Genussm.,  1904,  8,  433. 

ARACHIS  (PEANUT)  OIL. 

Sadtler:  Amer.  Journ.  Pharm.,  1897,  69,  490;  Analyst,  1897,  22, 
284. 

Archbutt:  Journ.  Soc.  Chem.  Ind.,  1898,  17,  1124. 

Perrin:  Monat.  scientif.,  1901,  [4],  15,  320;  Ztschr.  Unters.  Nahr.- 
Genussm.,  1901,  4,  986. 

Bellier  :  Bull.  Soc.  Chim.,  1902,  [3],  23,  358. 

Wijs :  Ztschr.  Unters.  Nahr.-Genussut.,  1903,  6,  692. 

COTTONSEED  OIL. 

Oilar :  Amer.  Chem.  Journ.,  1900,  24,  355. 

Weems  and  Grettenberg :  Proc.  Iowa  Acad.  Sci.,  1901  ;  Ztschr. 
Unters.  Nahr.-Genussm.,  1902,  5,  465. 
Fulmer  :  Journ.  Amer.  Chem.  Soc.,  1902,  24,  1148. 
Wijs:  Ztschr.  Unters.  Nahr.-Genussm.,  1903,6,  692. 
Milliau:  Compt.  rend.,  1904,  139,  807. 

Fischer  and  Peyau :  Ztschr.  Unters.  Nahr.-Genussm.,  1905,  9,  81. 
Emmett  and  Grindley :  Journ.  Amer.  Chem.  Soc.,  1905,  27,  263. 

SESAME  OIL. 

Bellier:  Ann.  Chim.  Analyt.,  1899,  4,  217;  Analyst,  1900, 
25,  SO. 

Bomer:  Ztschr.  Unters.  Nahr.-Genussm.,  1899,  2,  705. 

Wijs:  Ibid.,  1902,  5,  1150. 

Utz :  Chem.  Rev.  Fett  und  Harz  Ind.,  1902,  2,  177 ;  Abs.  Ztschr. 
Unters.  Nahr.-Genussm.,  1903,  6,  621. 

Kreis :  Chem.  Ztg.,  1903,27,  1030. 

Lehnkering :  Ztschr.  'bffentl.  Chem.,  1903,  9,  436;  Analyst,  1904, 
29,  96. 

MAIZE  OIL. 

Hopkins  :  Journ.  Amer.  Chem.  Soc.,  1898,  20,  948. 

Williams:  Analyst,  1900,25,  146. 

Vulte  and  Gibson:  Journ.  Amer.  Chem.  Soc.,  1900,22,  453  ; 
23,  I- 


FATTY    OILS— SPECIAL   METHODS.  165 

POPPYSEED    OlL. 

Utz:  Chem.  Ztg.,  1903,27,  1176. 

LINSEED  OIL. 

Gill  and  Lamb:  Jonrn.  Amer.  Chem.  Soc.,  1899,  21,  29. 

Wijs :  Chem.  Rev.  Fett  mid  Harz  Ind.,  1899,  6,  29;  Chem- 
Centrbl.,  1899,  I.,  646. 

Kitt:  Chem.  Rev.  Fett  und  Harz  Ind.,  1901,  8,  40;  Jonrn.  Soc* 
them.  Ind.,  1901,  20,  484. 

Mcllhiney:  Linseed  Oil  and  its  Adulterants,  New  York  State 
Dept.  Agriculture  (1901);  Sabin's  Technology  of  Paint  and  Var- 
nish, Chapter  V. 

Sjollema:  Ztschr.  Unters.  Nahr.-Genussm.,  1903,  6,  631. 

Lewkowitsch  :  Analyst,  1904,  29,  2. 

OXIDIZED  OILS. 

Ballantyne  :  Journ.  Soc.  Chem.  Ind.t  1891,  10,  29. 
Thompson  and  Ballantyne:  Ibid.,  1892,  n,  506. 
Fahrion :  Ztschr.  angew.  Chem.,  1898,  781. 
Lewkowitsch:  Analyst,  1902,  27,  139. 
Dunlap  and  Schenk :  Journ.  Amer.  Chem.  Soc.,  1903,  25,  826. 


CHAPTKR   XI 

Butter. 

Butter  is  officially  defined*  as  '  the  product  made  by  gathering 
in  any  manner  the  fat  of  fresh  or  ripened  cream  into  a  mass  which 
also  contains  a  small  portion  of  the  other  milk  constituents,  with 
or  without  salt.'  Standard  butter  contains  not  less  than  82.5 
per  cent,  of  butter  fat,  having  a  Reichert-Meissl  number  not  less 
than  24  and  a  specific  gravity  not  less  than  0.905  at  4O°/4O°. 
By  acts  of  Congress  approved  August  2,  1886,  and  May  9,  1902, 
butter  may  also  contain  additional  coloring  matter. 

Butter  may  fail  to  meet  the  requirements  of  the  official  standard 
either  because  of  a  deficiency  in  the  percentage  of  fat  or  because 
of  the  presence  of  foreign  fat  or  butter  fat  of  abnormal  character, 
though  the  sample  may  contain  nothing  which  can  be  regarded  as 
unwholesome.  Butter  analysis  therefore  includes  (i)  the  examina- 
tion of  the  whole  butter,  (2)  the  examination  of  the  butter  fat. 

This  chapter  will  be  devoted  mainly  to  the  methods  of  examin- 
ing butter  fat  but  for  convenience  the  determination  of  water,  fat, 
curd,  and  ash  will  be  described  first.  The  point  at  which  tests 
for  preservatives  can  conveniently  be  made  will  be  indicated,  but 
the  detection  of  foreign  colors  will  not  be  considered  as  these  are 
not  regarded  as  adulterants. 

DETERMINATION  OF  WATER,   FAT,   CURD,  AND  ASH. 

Especial  care  must  be  taken  in  sampling  butter  since  the  water, 
salt,  and  curd  are  often  unevenly  distributed  and  an  attempt  to  mix 
by  stirring  is  apt  to  result  in  squeezing  out  drops  of  brine.  If  a 
large  quantity  is  to  be  sampled  a  butter  trier  should  be  used,  and 
the  portions  thus  drawn  united  until  a  working  sample  of  about 
500  grams  is  obtained. 

Melt  the  sample  at  the  lowest  possible  temperature  in  a  wide- 
mouthed  glass-stoppered  bottle,  shake  violently  to  ensure  a  homo- 

*  Circular  No.  13,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 

1 66 


BUTTER.  1 6; 

geneous  mixture  and  continue  the  shaking  while  cooling  the 
sample  until  it  is  sufficiently  solidified  to  prevent  any  separation  of 
water  and  fat. 

Thoroughly  clean  and  dry  a  lipped  dish  or  beaker  having  a  flat 
bottom  of  at  least  20  square  centimeters.  Weigh  the  dish,  intro- 
duce 1.5  to  2  grams  of  butter  and  re-weigh  quickly  to  avoid  evap- 
oration. Dry  to  constant  weight  in  a  boiling  water  oven.  The 
loss  is  water.  Treat  the  dry  residue  with  petroleum  ether  or  ben- 
zine, transfer  it  to  a  weighed  Gooch  crucible  having  a  felt  of 
ignited  asbestos  and  wash  with  the  solvent  until  all  fat  is  removed. 
Dry  the  crucible  and  residue  to  constant  weight  in  a  water  oven 
or  an  air  bath  not  above  110°.  The  material  dissolved  by  the 
petroleum  ether  or  benzine  \sfat.  Burn  the  curd  at  a  temperature 
below  a  red  heat  and  weigh  the  crucible  containing  the  ash. 

Notes. —  The  method  given  is  essentially  that  of  the  Association 
of  Official  Agricultural  Chemists.*  If  preferred  the  butter  can  be 
dried  on  clean  dry  sand  or  asbestos.  When  the  latter  is  not  used 
it  is  important  that  the  butter  form  only  a  very  thin  layer  on  the 
bottom  of  the  dish  or  beaker ;  otherwise  the  water  sinks  into  the 
melted  fat  and  is  only  very  slowly  expelled  at  the  temperature  of 
the  boiling  water  oven.  For  convenience  in  transferring  and 
washing  the  residue  with  petroleum  ether  the  latter  should  be  used 
in  a  small  wash  bottle  having  a  ground  glass  stopper.  Instead  of 
drying  in  a  dish  and  transferring  the  residue  to  a  crucible,  the  but- 
ter may  be  weighed  directly  in  a  crucible  two-thirds  filled  with 
fibrous  asbestos,  dried  to  constant  weight  and  then  extracted.f  A 
device  for  facilitating  this  drying  by  passing  a  current  of  air  through 
the  heated  crucible  has  recently  been  described  by  Bird.J 

Great  care  must  be  taken  to  burn  the  curd  at  the  lowest  tem- 
perature possible  in  order  to  avoid  loss  of  chlorine  (see  Chapter  I.). 
Any  small  amount  of  milk  sugar  which  the  butter  might  con- 
tain would  be  counted  as  curd  in  this  analysis.  The  percentage 
of  salt  can  be  found  by  determining  chlorine  in  the  ash,  or  by 
repeatedly  washing  the  butter  with  hot  water  in  a  separatory 
funnel  and  titrating  the  combined  washings  with  a  standard  solu- 
tion of  silver  nitrate.  Good  butter  usually  contains  10  to  14  per 
cent,  water,  84  to  87  per  cent,  fat,  0.5  to  1.5  per  cent,  curd,  2.0  to 

*Bul.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

f  Richards  and  Woodman  :  Air,  Water,  and  Food  (2d  Ed.),  p.  2OI. 

\Journ.  Amer.  Chem.  Soc.,  1905,  27,  818. 


1 68  ORGANIC   ANALYSIS. 

4.0  per  cent,  ash  if  salted;  if  unsalted,  0.25  to  0.5  per  cent.  The 
water  content  of  butter  has  often  been  limited  by  legal  or  trade 
standards  to  16  per  cent.  This  is  considerably  above  the  present 
average  for  creamery  butter,  as  shown  by  an  investigation  made 
by  the  United  States  Department  of  Agriculture  in  1902.*  Of  800 
samples  from  400  creameries  in  18  states,  the  average  water  con- 
tent was  11.78  percent;  85  per  cent,  of  the  samples  contained 
between  10  and  14  per  cent. ;  the  extreme  limits  were  7.20  and  17.62 
per  cent.  The  amount  of  salt  added  to  butter  varies  greatly  with 
the  demands  of  different  markets,  but  over  5  percent,  would  be  ex- 
cessive unless  the  butter  were  intended  for  export  to  a  tropical 
country.  An  excessive  amount  of  curd  indicates  careless  manu- 
facture or  fraudulent  increase  of  weight  and  is  likely  to  injure  the 
keeping  qualities  of  the  butter. 

EXAMINATION  OF  BUTTER  FAT. 

PREPARATION. 

Melt  100  grams  or  more  of  the  butter  and  allow  it  to  stand  at 
45°  to  55°  until  the  water  and  salt  settle  to  the  bottom.f  Pour 
off  the  melted  fat  by  decantation  and  filter  it  through  a  dry  paper 
in  a  funnel  heated  by  a  water  jacket  or  supported  in  a  drying  oven 
kept  at  about  60°.  The  filtered  fat,  which  must  be  free  from  tur- 
bidity, is  received  in  a  wide-mouthed  bottle  and  kept  stoppered 
in  a  cool  place  until  analyzed. 

REICHERT.MEISSL  OR  REICHERT.WOLLNY  NUMBER.^ 
This  number  is  a  comparative  measure  of  the  proportion  of  vol- 
atile acids,  and  is  the  most  important  basis  for  deciding  the  purity 
of  butter  fat.  Often  the  presence  or  absence  of  foreign  fat  in  butter, 
or  the  proportion  of  butter  fat  in  oleomargarine,  is  inferred  from  this 
number  alone.  In  the  interest  of  uniformity  of  results  official  chem- 

*  Circular  No.  39,  Bureau  of  Animal  Industry  ;  Analyst,  1903,  28,  184. 

|  This  water  solution  can  be  tested  for  preservatives,  of  which  boric  acid  and  borax 
are  most  likely  to  be  found  in  butter.  For  methods  see  the  chapter  on  milk  analysis, 
or  for  more  detailed  directions  Leach's  Food  Inspection  and  Analysis,  or  Lewkowitsch's 
Oils,  Fats,  and  Waxes. 

JReichert:  Ztschr.  anal.   Chem.,  1879,  18,  69. 

Meissl:   Dingier' s  Polytech.  Journ.,  1879,  233,  229. 

Wollny :  Milch  Ztg.,  1887,  16,  609  ;  Analyst,  1887,  12,  203,  235  ;   1888,  13,  8,  38. 

These  papers  are  reprinted  in  Ephraim's  Original  Arbeiten  iiber  Analyse  der  Nah- 
rungsmittel. 


BUTTER.  169 

ists  have  sought  to  describe  the  method  in  such  detail  as  to  elim- 
inate variations  due  to  manipulation. 

Reagents.  —  I.  Caustic  soda  solution,  made  by  dissolving  sodium 
hydroxide  (nearly  free  from  carbonate)  in  an  equal  weight  of  dis- 
tilled water. 

2.  Alcohol,  92  to  95  per  cent,  containing  no  appreciable  amount 
of  volatile  acid,  either  free  or  combined. 

3.  Dilute  sulphuric  acid,  made  by  mixing  pure  concentrated  sul- 
phuric acid  with  four  times  its  volume  of  water. 

4.  An  accurately  standardized  approximately  tenth-normal  solu- 
tion of  barium  (or  sodium)  hydroxide. 

5.  A  i  per  cent,  solution  of  phenolphthalein  in  alcohol. 
Determination.  —  Thoroughly   clean  and  dry  a  flask  of  250  to 

300  c.c.  capacity.  Weigh  the  flask,  thoroughly  mix  the  melted  fat, 
introduce  5.6  to  5.8  c.c.  measured  at  about  5°°»  allow  the  flask  and 
fat  to  cool  for  15  to  20  minutes  and  re-weigh  (or,  if  convenient, 
weigh  exactly  5  grams  of  fat  into  the  flask).  Add  10  c.c.  of  the 
alcohol  and  2  c.c.  of  the  caustic  soda  solution,  attach  the  flask  to'a 
reflux  condenser  and  boil  on  a  water  bath  or  electric  heater  for  at 
least  half  an  hour  to  ensure  complete  saponification.* 

Evaporate  the  alcohol  by  heating  the  flask  in  a  steam  bath,  shak- 
ing occasionally  to  avoid  danger  of  loss  from  frothing  and  to  facil- 
itate the  removal  of  the  alcohol.  Add  1 32  c.c.  of  recently  boiled 
distilled  water,  warm  at  60°  to  70°  until  the  soap  is  completely 
dissolved,  add  8  c.c.  of  the  dilute  sulphuric  acid  and  a  few  pieces 
of  pumice  stone,  re-stopper  the  flask  or  connect  it  with  a  condenser 
and  warm  without  boiling  until  the  fatty  acids  separate  as  a  clear 
layer.  Distil  through  a  glass  condenser,  collecting  the  distillate 
in  a  flask  accurately  graduated  at  no  c.c.  The  distillation  should 
be  so  regulated  that  no  c.c.  will  be  collected  in  from  28  to  32 
minutes.  Mix  the  distillate,  filter  through  dry  paper,  and  titrate 
100  c.c.  of  the  filtrate,  using  0.5  c.c.  of  the  phenolphthalein  solu- 
tion as  indicator,  until  the  red  color  remains  apparently  un- 
changed for  2  minutes.  Increase  the  burette  reading  by  one  tenth 
(on  account  of  the  10  c.c.  of  distillate  not  titrated),  and  calculate 
the  number  of  cubic  centimeters  of  tenth-normal  alkali  which 
would  have  been  required  if  exactly  five  grams  of  fat  were  taken 
for  the  determination.  This  is  the  Keichert-Meissl  number. 

*The  saponification  can  also  be  accomplished  by  heating  in  a  closed  flask,  using 
either  aqueous  or  alcoholic  alkali,  or  by  means  of  the  glycerol-soda  solution  pro- 
posed by  Leffmann  and  Beam:  Analyst,  1891,  16,  153. 


i/o  ORGANIC   ANALYSIS. 

Notes. — The  number  thus  found  does  not  represent  the  total  vol- 
atile acids  present.  The  yield  is  fairly  uniform  if  the  given  condi- 
tions of  dilution  and  distillation  are  maintained.  Wollny  submitted 
this  method  to  an  exhaustive  examination  and  pointed  out  the  fol- 
lowing sources  of  error:  (i)  absorption  of  carbon  dioxide  during 
saponification,  (2)  formation  of  esters  during  saponification,  (3)  for- 
mation of  esters  during  distillation,  (4)  coherence  of  fatty  acids  dur- 
ing distillation  resulting  in  holding  back  some  of  the  volatile  acid, 
(5)  variations  in  the  proportion  of  volatile  acid  carried  over,  due 
to  differences  in  size  and  shape  of  distillation  apparatus.  In  order  to 
avoid  discrepancies  from  these  and  other  causes  he  published  an 
elaborately  detailed  system  of  manipulation  and  precautions.  It  has 
been  shown  that  Wollny  greatly  overestimated  the  probable  errors 
of  the  method  as  previously  carried  out  and  that  some  of  his  precau- 
tions are  unnecessary,  but  as  in  the  main  they  tend  toward  greater 
uniformity  of  results,  they  have  been  adopted  with  slight  modifi- 
cations by  the  Association  of  Official  Agricultural  Chemists,  whose 
methods  *  are  usually  accepted  as  standard  in  the  United  States  and 
should  be  followed  exactly  in  any  determination  which  is  likely  to 
be  made  the  basis  of  legal  action. 

In  Great  Britain,  a  joint  committee  representing  the  Govern- 
ment Laboratory  and  the  Society  of  Public  Analysts  has  adopted 
the  method  essentially  as  described  above  with  the  following 
specifications  for  the  apparatus  to  be  employed  :  f  Flask  used  for 
saponification  and  distillation;  capacity,  300  c.c.;  length  of  neck, 
7  to  8  cm. ;  width  of  neck,  2  cm.  The  flask  is  connected  with  the 
condenser  by  means  of  a  bent  glass  tube  7  mm.  wide,  so  placed 
that  the  bend  is  1 5  cm.  above  the  top  of  the  cork.  At  a  distance 
of  5  cm.  above  the  cork  is  a  bulb  5  cm.  in  diameter.  The  flask  is 
supported  on  a  circular  piece  of  asbestos  12  cm.  in  diameter,  hav- 
ing a  hole  5  cm.  in  diameter  in  the  center,  so  that  the  bottom  of 
the  flask  is  heated  by  a  free  flame  during  the  distillation.  The 
British  committee  further  prescribed  that  blank  determinations  be 
made  and  the  volume  of  alkali  found  necessary  to  neutralize  the 
distillate  (which  volume  should  not  exceed  0.3  c.c.)  be  deducted  in 
calculating  the  results  of  each  determination.  The  number  so 
obtained  is  called  the  Reichert- Wollny  number. 

The  Reichert-Meissl  or  Reichert- Wollny  number  of  butter  fat  is 

*  Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture, 
f  Analyst,  1900,  25,  309. 


BUTTER.  171 

usually  between  24  and  34;  that  of  cocoanut  fat,  between  6  and  8; 
of  other  edible  fats  and  oils,  usually  less  than  I. 

SPECIFIC  GRAVITY. 

The  specific  gravity  of  butter  fat  has  often  been  determined  either 
at  1 00°  or  at  37.8°  (100°  F.).  The  standard  recently  established 
for  the  United  States  prescribes  a  minimum  specific  gravity  at  40°, 
water  at  the  same  temperature  being  taken  as  unity.  Either  a 
specific  gravity  flask  or  an  Ostwald  pyknometer  can  be  used  con- 
veniently for  the  determination,  the  pyknometer  being  filled  and 
adjusted  while  surrounded  by  water  kept  at  the  required  tempera- 
ture, then  removed  from  the  water  bath,  wiped  dry  on  the  out- 
side, allowed  to  cool  to  the  temperature  of  the  balance  and  weighed. 

SAPONIFICATION  NUMBER. 

This  is  determined  as  described  in  Chapter  IX.  Since  the  normal 
saponification  numbers  of  butter  fat  are  only  about  15  per  cent,  in 
excess  of  those  of  the  fats  commonly  used  as  adulterants,  the  de- 
termination in  order  to  be  of  much  value  must  be  very  accurately 
made. 

INSOLUBLE  FATTY  ACIDS — HEHNER  NUMBER. 

Reagents. —  i.  The  alcoholic  potash  solution  used  in  the  deter- 
mination of  the  saponification  number. 

2.  Alcohol,  about  95  per  cent,  by  volume,  which  leaves  no  ap- 
preciable residue  upon  evaporation. 

Determination.  —  Saponify  4  grams  of  butter  fat  with  50  c.c.  of 
the  alcoholic  potash  solution,  evaporate  to  a  paste  to  expel  alcohol, 
dissolve  the  soap  in  about  400  c.c.  of  hot  water  in  a  weighed 
beaker,  add  hydrochloric  acid  in  excess  of  the  amount  required  to 
neutralize  the  potash  used,  and  heat  nearly  to  boiling  with  occa- 
sional stirring  until  the  fatty  acids  have  collected  in  a  clear  layer  on 
the  surface.  Cool  thoroughly,  pour  the  solution  through  a  filter 
and  wash  the  cake  with  cold  water  without  removing  it  from  the 
beaker.  Stir  up  the  fatty  acids  in  the  beaker  with  another  portion 
of  hot  water  (200  to  300  c.c.),  cool  thoroughly,  filter,  and  wash 
again.  Repeat  this  treatment  three  times.  After  a  final  thorough 
washing  with  cold  water,  put  the  beaker  containing  the  fatty  acids 
beneath  the  funnel  and  dissolve  any  fatty  acids  which  the  filter  may 
contain  by  washing  with  hot  95  per  cent,  alcohol,  allowing  the 
washings  to  run  into  the  beaker.  Evaporate  off  the  alcohol  and 


172  ORGANIC   ANALYSIS. 

dry  the  beaker  containing  the  fatty  acids  to  constant  weight  in  a 
boiling  water  oven. 

Notes.  —  This  is  the  modification  of  Hehner's  method  adopted 
by  the  Association  of  Official  Agricultural  Chemists  *  and  by  the 
chemists  of  the  Government  Laboratory,  London.f  The  original 
method  J  which  is  still  largely  used  involves  washing  the  melted 
fatty  acids  with  hot  water  on  a  paper  filter.  The  results  thus  ob- 
tained are  usually  I  to  2  per  cent,  lower  than  those  by  the  official 
method.  In  the  hot  filtration  method  there  is  danger  of  washing 
some  of  the  melted  fatty  acid  through  the  paper. 

IODINE  NUMBER. 

The  determination  of  the  iodine  number  has  been  fully  described 
in  Chapter  IX.  As  butter  fat  absorbs  only  26  to  38  per  cent,  of  its 
weight  of  iodine,  a  gram  of  sample  can  be  used  for  each  determi- 
nation. According  to  Patrick  §  butter  fat  shows  iodine  numbers 
about  I  unit  higher  by  the  Hanus  than  by  the  Hubl  method. 

MELTING  POINT — WILEY'S  METHOD.|| 

Apparatus  and  Reagents.  —  I.  An  accurate  thermometer  reading 
to  o.i  degree. 

2.  A  tall  beaker  nearly  filled  with  water  and   arranged  to  be 
heated  gradually  with  constant  stirring  from  bottom  to  top. 

3.  A  wide  test  tube  suspended  in  the  water  in  the  beaker  and 
nearly  filled  with  water  and  alcohol  as  follows  :   Half  fill  the  tube 
with  hot  recently  boiled  distilled  water,  then  pour  a  nearly  equal 
volume  of  hot  recently  boiled  alcohol  into  the  tube,  carefully  float- 
ing the  alcohol  on  the  water  with  as  little  mixing  of  the  liquids  as 
possible. 

Determination.  —  Allow  a  drop  of  the  melted  butter  fat  to  fall 
upon  a  smooth  piece  of  ice  floating  in  recently  boiled  distilled 
water.  A  thin  disc  of  fat  about  I  cm.  in  diameter  should  be  ob- 
tained. Remove  the  disc  from  the  ice  by  forcing  the  latter  below 
the  water  when  the  fat  will  come  to  the  surface  whence  it  is 

*Bul.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

\  Thorpe  :  Journ.  Chem.  Soc.,  1904,  85,  248. 

J  Hehner  and  Angell:  Butter,  its  Composition  and  Adulterations.  London,  1874. 
Hehner  :  Ztschr.  anal.  Chem.,  1877,  16,  145.  Ephraim,  loc.  cit. 

\  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  49. 

||  Wiley's  Agricultural  Analysis,  Vol.  III.;  Bui.  46,  Bur.  Chem.,  U.  S.  Dept. 
Agriculture. 


BUTTER.  1/3 

removed  by  means  of  a  steel  spatula  or  knife  blade  and  dropped 
into  the  tube  containing  the  water  and  alcohol.  The  disc  sinks  to 
the  point  where  the  density  of  the  alcohol-water  mixture  is  equal 
to  its  own.  It  must  not  touch  the  side  of  the  tube.  Suspend  the 
thermometer  so  that  the  bulb  hangs  in  the  tube  exactly  level  with 
the  disc  of  fat.  Gradually  heat  the  beaker,  keeping  the  water  well 
stirred.  After  the  disc  begins  to  shrivel,  indicating  that  the  tem- 
perature is  within  a  few  degrees  of  the  melting  point,  the  heat 
must  be  applied  very  carefully.  The  temperature  at  which  the  fat 
becomes  a  sphere  is  taken  as  the  melting  point.  Repeat  the 
determination  twice,  heating  the  bath  at  such  a  rate  that  8  to  10 
minutes  are  required  to  raise  the  temperature  through  the  last 
2  degrees.  The  second  and  third  determinations  should  agree 
within  0.2  degree. 

Notes. —  The  special  advantage  of  this  method  is  that  it  avoids 
the  discrepancies  caused  by  the  adherence  of  the  melting  fat  to 
solid  surfaces,  which  in  most  other  methods  makes  it  difficult  to 
determine  the  exact  point  of  fusion.  It  is  important  to  secure  a 
very  thin  disc  of  fat  for  the  determination,  to  avoid  all  adherence 
of  air  bubbles,  and  to  secure  uniform  heating  of  the  thermometer 
bulb  and  the  disc  by  occasionally  swaying  the  former  around  the 
latter  as  the  temperature  approaches  the  melting  point.  By  using 
hot  recently  boiled  water  and  alcohol  in  preparing  the  test  tube  for 
the  determination  the  danger  of  air  bubbles  is  avoided. 

ADDITIONAL  DETERMINATIONS. 

The  index  of  refraction  and  Crismer's  test*  based  upon  the  critical 
temperature  of  solution  in  alcohol  are  considerably  used  in  the 
examination  of  butter  fat.  They  are  especially  useful  where  rapid 
"  sorting  tests "  are  required,  as  in  food  inspection  laboratories 
where  only  the  suspected  samples  are  submitted  to  complete 
examination. 

The  phytosteryl  acetate  test\  is  occasionally  employed  as.a  means 
of  detecting  the  presence  of  vegetable  fat,  but  requires  too  much 

*Crismer:    Bull.  Assoc.  beige.  Chirn.,  1895,  9,  71  ;   1896,  10,  312;    Abs.  Analyst, 

1895,  20,  209  ;  1897,  22,  71.    Seealso — Weiss  :  Pharm.  Ztg.,  41,268  ;  Chem.  Centrbl., 

1896,  I.,  1212.    Asboth:   Chem.  Ztg.,  1896,  20,  685.    Browne:  Jonrn.  Amer.  Chem. 
Soc.,  1899,  21,  990.     Lewkowitsch  :  Oils,  Fats,  and  Waxes  (3d  Ed.),  214,  86l. 

fBoiner:  Ztschr.  Unters.  Nakr.-Genussm.,  1901,  4,  865,  1070;  1902,5,  1018. 
Juckenack  and  Pasternack  :  Ibid.,  1904,  7,  193.  Lewkowitsch  :  /.  c.,  373. 


i/4  ORGANIC  ANALYSIS. 

time  and  skill  for  ordinary  use  and  is  likely  to  give  misleading 
results  in  the  hands  of  an  inexperienced  person.  Cocoanut  oil  is 
the  only  vegetable  fat  which  could  be  used  in  any  considerable 
quantity  without  being  shown  by  the  Reichert-Meissl  number,  and 
the  presence  of  cocoanut  oil  is  best  shown  by  comparison  of  the 
Reichert-Meissl  and  saponification  numbers  or  determination  ot 
the  insoluble  volatile  acids  as  described  later. 

Acidity  of  butter  fat  is  sometimes  determined  and  interpreted  as 
a  measure  of  the  rancidity,  although  the  odor  and  taste  which 
cause  a  butter  to  be  regarded  as  rancid  are  more  largely  due  to 
aldehydes  and  other  decomposition  products  than  to  free  fatty 
acids.  On  the  assumption  that  acidity  can  serve  as  a  measure 
of  rancidity  the  term  degree  of  rancidity  is  sometimes  used  as 
synonomous  with  degree  of  acidity,  i.  e.,  to  show  the  number  of 
c.c.  of  normal  alkali  required  to  neutralize  the  free  acid  in  IOO 
grams  of  fat.  One  "  degree  "  is  therefore  equivalent  to  an  acid 
number  of  0.56  or  to  0.28  per  cent,  of  free  oleic  acid. 

COMPOSITION  OF  BUTTER  FAT. 

Browne  analyzed  the  mixture  of  fatty  acids  from  a  sample 
having  a  rather  low  iodine  number  (29.28)  with  the  following 
results :  — * 

Percentage  of        Corresponding  Percentage 
Acids.  Acid  in  Fat.  of  Triglyceride. 

Oleic 32.50  33.95 

Dioxysiearic i.oo  1.04 

Stearic 1.83  1.91 

Palmitic 38.61  40.51 

Myr-istic 9.89  10.44 

Laurie .• 2.57  2.73 

Capric 0.32  0.34 

Caprylic 0.49  0.53 

Caproic 2.09  2.32 

Butyric 5.45  6.23 

Total 94-75  100.00 

This  calculation  neglects  the  unsaponifiable  matter,  which  ac- 
cording to  Browne  amounts  to  only  about  o.i  per  cent. 

The  composition  of  butter  fat  is,  however,  quite  variable  as  will 
be  seen  from  the  range  in  analytical  properties. 

* /ourn.  Amer.  Chem.  Soc.,  1899,  21,  823. 


BUTTER. 


175 


VARIATIONS  AND  RELATIONS  OF  ANALYTICAL  PROPERTIES. 

The  Reichert-Meissl  or  Reichert-Wollny  number  is  much  the 
most  important  of  the  data  obtained  in  the  examination  of  butter 
fat.  The  proportion  of  volatile  acids  tends  to  decrease  as  the 
period  of  lactation  advances.  The  estimated  normal  range  for  the 
other  important  properties  has  been  given  in  the  table  at  the  end 
of  Chapter  IX. 

Any  of  these  properties  may  be  influenced  by  the  feeding  or 
health  of  the  animal  and  occasionally  vary  much  beyond  the  usu- 
ally accepted  "  normal  "  limits  as  is  shown  by  the  following  data 
collected  by  Browne.* 

General  Limits.  Extreme  Limits. 

Reichert-Meissl  number  .........       20-33         n.2[Morse]-4i[Nilson] 

Saponification  number  ...........  220-236         2i6[Samelson]-245  [Fischer] 

Iodine  number  ....................       26-38         i9.5[Moore]-49.57 


The  results  of  analyses  of  357  authentic  samples  of  butter  fat 
collected  from  various  parts  of  Great  Britain  and  examined 
at  the  Government  Laboratory,  London,  have  been  arranged  by 
Thorpe  t  according  to  the  Reichert-Wollny  numbers  and  averaged 
by  groups  to  show  the  relations  between  the  principal  physical 
and  chemical  properties  of  pure  butter  fat. 

The  following  table  shows  the  averages  for  each  group  of  sam- 
ples. The  first  line,  for  example,  gives  the  average  Reichert- 
Wollny  number  for  all  samples  in  which  this  number  lay  between 


RELATION  OF  PHYSICAL  AND  CHEMICAL  PROPERTIES  OF  BUTTER  FAT  (THORPE). 


Number 
of 
Samples. 

Reichert- 
Wollny 
Number. 

Specific 
Gravity 
37-8° 
37& 

Butyro- 
Refractometer 
Reading 
at  45°. 

Saponification 
Number. 

Insoluble 
Fatty  Acids 
Per  Cent. 

Mean 
Molecular 
Weight  of 
Insoluble 
Acids. 

7 

22.5 

0.9101 

42.O 

219.9 

90.1 

266.9 

17 

23-5 

0.9104 

41-5 

221.6 

89.7 

265.5 

15 

24-5 

0.9108 

41.5 

223.5 

89.4 

265.0 

27 

25-5 

0.9110 

41-3 

223.6 

89.3 

264.2 

37 

26.5 

0.9113 

4LO 

225.6 

88.9 

261.9 

5i 

27-5 

0.9114 

40.6 

227.0 

88.7 

261.7 

78 

28.8 

0.9118 

40.1 

228.6 

88.4 

260.9 

56 

29.5 

0.9120 

40.1 

230.2 

88.3 

259.6 

41 

30-5 

0.9123 

39-9              231.7 

87.9 

260.1 

18 

31.3 

0.9125 

39-7 

232.5 

87.9 

258.0 

10 

32.6 

0.9130 

39-4 

232.8 

87.7 

257.8 

*Journ.  Amer.  Chem.  Soc.,  1899,  21,  632. 
^Journ.  Chem.  Soc.,  1904,  85,  248. 


ORGANIC  ANALYSIS. 


22.00  and  22.99,  with  the  average  of  the  same  samples  for  each  of 
the  other  determinations.  The  second  line  shows  the  averages  for 
all  samples  having  Reichert-Wollny  numbers  from  23.00  to  23.99, 
etc. 

In  order  to  show  to  what  extent  increase  of  volatile  acids  takes 
place  at  the  expense  of  oleic  acid,  the  iodine  numbers  of  50  of  the 
above  samples  were  determined.  Arranging  these  in  groups  of  20 
and  30  respectively,  according  to  the  Reichert-Wollny  values,  the 
following  average  figures  were  obtained  :  — 


Reichert- 
Wollny 
Number. 

Iodine 
Number. 

Oleic 
Acid 
Per  Cent. 

Insoluble 
Acid 
Per  Cent. 

Mean  Molec- 
ular Weight  of 
Insoluble  Acids. 

First  group, 

24.2 

40.0 

44-4 

89.6 

264.6 

Second  group, 

30.8 

32-4 

36.0 

88.1 

259.8 

DETECTION  OF  OLEOMARGARINE. 

Butter  substitutes  or  "  artificial  butters,"  unless  sold  under  special 
names  indicating  their  origin,  are  collectively  termed  "  oleomarga- 
rine "  in  America  or  "  margarine  "  in  England.  The  oleomargarine 
made  in  America  *  consists  chiefly  of  refined  lard,  "  oleo  oil"  (the 

*  The  materials  used  in  the  manufacture  of  oleomargarine  in  the  United  States  dur- 
ing the  fiscal  year  ending  June  30,  1899  (Senate  Document  No.  168,  57th  Congress, 
1st  Session)  were  as  follows: 


Material. 

Quantity. 

Percentage 
of  the 
whole. 

Value 
per 

pound. 

Total  value. 

Neutral  lard 

Pounds. 
3I,2Q7,2CI 

34.27 

Cents. 
8 

$2,503,780  08 

Oleo  oil.  ... 

24.401,760 

26.82 

2,I44,QI7  60 

Cotton-seed  oil. 

4,-5C7,Cl4 

4  77 

6 

522,025  08 

"  Butter  oil  ".. 

4,742,QO4 

4.76 

6 

260,520.00 

Sesame  oil  .  . 

486,310 

53 

10 

4,863.10 

Coloring  matter  

148,970 

.16 

20 

2O,2q6.OO 

Sugar  

IIO,l64 

.12 

4 

4,406.  no 

Glycerin  

8,963 

.OI 

10 

896.30 

Stearin  

15,890 

!.oo7 

8 

4^9.60 

Glucose  

2,550 

.003 

3 

76.50 

Milk  

14,200,576 

IC.CC 

I 

142,005.76 

Salt  

6,77^,670 

7.42 

I 

67,726.70 

Butter 

1,568,310 

I   72 

20 

313,663.80 

Cream 

3,527,410 

3  86 

c 

176,370.  i»o 

Total  

91,322,260 

100 

6,171,007.61 

"  Butter  oil  "  is  commonly  stated  to  be  a  special  brand  of  cottonseed  oil ;  but  the 
high  Federal  tax  laid  upon  artificially  colored  oleomargarine  by  the  law  of  May  9, 
1902,  practically  prohibits  the  use  of  coloring  matters  employed  before  that  date  and 
has  led  to  the  introduction  of  "butter  oils"  containing  palm  oil  which  is  naturally 
highly  colored  (Crampton  and  Simons  :  Journ.  Amer.  Chem.  Soc.,  1905,  27,  270). 


BUTTER.  177 

soft  part  of  beef  fat),  and  cottonseed  oil,  often  mixed  with  a  small 
amount  of  butter  and  almost  always  churned  with  milk  or  cream. 
Palm  oil  and  sesame  oil  are  known  to  be  used  to  some  extent  and 
other  semi-drying  and  non-drying  oils  are  probably  utilized  in 
some  factories. 

Hence  in  comparison  with  any  of  the  common  constituents  of 
oleomargarine,  butter  fat  is  characterized  by  its  high  proportion 
of  soluble  volatile  acids,  together  with  a  high  percentage  of  lauric, 
myristic,  and  palmitic  as  compared  with  oleic  and  stearic  acids. 
The  presence  of  oleomargarine  in  butter  fat,  therefore,  lowers  the 
Reichert-Meissl  and  saponification  numbers  and  the  specific 
gravity,  while  it  raises  the  percentage  of  insoluble  acids  and  either 
the  melting  point  or  the  iodine  number  or  both. 

Several  European  governments  require  that  sesame  oil  be  added 
to  oleomargarine  in  order  to  facilitate  its  detection  when  mixed 
with,  or  substituted  for,  butter.  Similarly  the  addition  of  butter 
to  oleomargarine  is  sometimes  forbidden  or  restricted  in  order  to 
prevent  the  production  of  mixtures  too  closely  resembling  genuine 
butter.  The  essential  features  of  the  oleomargarine  laws  of  the 
principal  countries  are  given  in  Lewkowitsch's  Oils,  Fats,  and 
Waxes. 

DETECTION  OF  COCOANUT  FAT. 

Since  cocoanut  fat  consists  largely  of  glycerides  of  saturated  acids 
of  low  molecular  weight,  it  could  be  added  in  considerable  quan- 
tity to  butter  fat  of  average  composition  without  causing  the 
latter  to  vary  beyond  the  normal  limits  in  any  of  the  important 
analytical  properties.  A  comparison  of  the  Reichert-Meissl  and 
saponification  numbers,  however,  would  lead  to  the  detection  of 
this  adulteration  since  the  former  number  is  higher  and  the  latter 
lower  in  butter  than  in  cocoanut  fat.  In  pure  butter  fat  the  value 
of  the  factor  [Saponification  number  —  (200+  Reichert-Meissl  num- 
ber)] varies  from  3.4  to  —  4.1  ;  in  pure  cocoanut  fat  it  varies  from 
47  to  50.7.* 

Another  method  of  showing  the  presence  of  cocoanut  oil  is  to 
determine  the  volume  of  tenth-normal  alkali  required  to  neutralize 
the  insoluble  volatile  acids  from  5  grams  of  fat.  Under  the  condi- 
tions described  by  Polenskef  the  results  thus  obtained  are  approx- 

*  Juckenack  and  Posternack  :  Ztschr.  Unters.  Nakr.-Genussm.,  1904,  7,  193. 

tPolenske  :  Ibid.,  1904,  7,  273.  See  also  — Lewkowitsch's  Oils,  Fats,  and  Waxes 
(3d  Ed.),  853.  Muntz  and  Coudon  :  Ann.  d.  Inst.  Agron.,  1904  ;  Analyst,  1905,  30, 
155.  Hesse :  Milchwirthschaftl.  Centrbl.,  1905,  i,  13;  Chem.  Centrbl.,  1905,  I.,  566. 


1 78  ORGANIC  ANALYSIS. 

imately  quantitative,  the  percentage  of  insoluble  volatile  acids 
(mainly  lauric  acid)  being  much  higher  in  cocoanut  fat  than  in 
butter. 

ADDITIONAL  REFERENCES. 

Behrend  and  Wolfs  :  Untersuchung  liber  die  Zusammensetzung 
und  die  Beschaffenheit  des  Butterfettes  aus  der  Milch  einzelner 
Kline,  Ztschr.  Unters.  Nahr.-Genussm.,  1902,  5,  689. 

Browne  :  The  Chemistry  of  Rancidity  in  Butter-fat,  Journ.  Amer. 
Chem.  Soc.y  1899,  21,  975. 

Cochran :  Butter  and  Butter  Adulterants,  Journ.  Franklin  Inst., 

1899,  147,  85. 

Crampton :  The  Composition  of  Process  or  Renovated  Butter, 
Journ.  Amer.  Chem.  Soc.y  1903,  25,  358. 

Crampton  and  Simons  :  The  Detection  of  Palm  Oil  when  used 
as  a  Coloring  Material  in  Oils  and  Fats,  Journ.  Amer.  Chem.  See., 
1905,  27,  270. 

Estcourt:  Butters  from  Various  Countries  Compared,  Analyst, 

1900,  25,  113. 

Grossmann  and  Meinhard  :  Zur  Beurtheilung  der  hollandischen 
Butter,  Zlschr.  Unters.  Nahr-Genussm.,  1904,  8,  237. 

Holm,  Krarup,  and  Petersen  :  Untersuchungen  liber  das  Licht- 
brechungsvermogen,  die  Jodzahl,  und  den  Gehalt  des  Butterfettes 
an  fliichtigen  Fettesauren,  Abs.  Ztschr.  Untesr.  Nahr.-Genussm., 

1901,4,  746. 

Jensen  :  Studien  liber  das  Ranzidwerden  der  Butter,  Centrbl. 
Bakteriol.,  II.  Abth.,  1902,  8  ;  Abs.  Ztschr.  Untesr.  Nahr.-Genussm., 

1903,  6,  376. 

Kirsten  :  Beitrage  zur  Untersuchung  und  Kenntniss  der  Zusam- 
mensetzung der  Milchfettes.  I.  Die  unverseifbare  Substanz  des 
Milchfettes,  Ztschr.  Unters.  Nahr.-Genussm.,  1902,  5,  833. 

Konig :  Chemie  der  Menschen  Nahrungs-  und  Genussmittel, 
(4th  Ed.),  Berlin,  1904. 

Kraus :  Untersuchungen  liber  den  Einfluss  der  Herstellung, 
Verpackung  und  Kochsalzgehaltes  der  Butter  auf  ihre  Haltbarkeit, 
Arb.  Kaiserl.  Gesundheitsamt.,  1904,  235  ;  Ztschr.  Unters.  Nahr.- 
Genussm.,  1905,  9,  286. 

Laxa  :  Ueber  die  Spaltung  des  Butterfettes  durch  Mikroorgan- 
ismen,  Arch.  Hygiene,  1902,  41,  119. 

Partheil  and  Velsen :  Die  Grundlagen  der  refraktometrischen 
Butteruntersuchung,  Arch.  Pharm.,  1900,  238,  261 ;  Abs.  Ztschr. 
Unters.  Nahr.-Genussm.,  1901,  4,  454. 


BUTTER.  179 

Patrick  :  Household  Tests  for  the  Detection  of  Oleomargarine 
and  Renovated  Butter,  Farmer's  Bulletin  No.  131,  U.  S.  Dept. 
Agriculture. 

Richmond:  Dairy  Chemistry.     London,  1899. 

Rogers :  Studies  upon  the  Keeping  Quality  of  Butter,  I.  Canned 
Butter,  Bui.  57,  Bur.  Animal  Industry,  U.  S.  Dept.  Agriculture. 

Siegfeld :  Zur  Beurtheilung  der  Butter  auf  Grund  der  Reichert- 
Meissl'schen  Zahl,  Ztschr.  Untcrs.  Nahr.-Genussm.,  1901,  ,  433, 
and  Molk.  Ztg.y  1904,  18,  481. 

Stebbins :  The  Reichert  Number  of  Butter,y<?#r#.  Amer.  Chem. 
Soc.,  1899,  21,938. 

Stohmann  :  Milch-  und  Molkereiproducte.     Braunschweig,  1898, 

Swaving :  Ueber  den  Einfluss  der  Baumwollsamenmehl-  und 
Sesamkuchen-Futterung  auf  die  Beschaffenheit  des  Butterfettes, 
Ztschr.  Unters.  Nahr.-Genussm.,  1903,  6,  97. 

Wiley:  U.  S.  Dept.  Agriculture,  Div.  Chem.,  Bui.  13,  Part  I, 
(1887).  Agricultural  Analysis,  Vol.  III.,  Easton,  Penn.,  1897. 


CHAPTER  XII. 

Soaps  and  Lubricants. 

Soaps,  glycerol,  and  lubricants  are  the  most  important  technical 
products  of  the  fats  and  oils.  Glycerol  having  been  considered 
under  the  head  of  alcohols,  methods  for  the  examination  of  soaps 
and  lubricants  will  be  given  in  this  chapter.  Any  compound  of  a 
mineral  base  with  a  fatty  or  resin  acid  maybe  called  a  soap.  Since 
only  the  soaps  of  the  alkalies  are  readily  soluble  in  water,  they 
alone  are  employed  as  detergents  but  insoluble  soaps  are  fre- 
quently used  as  constituents  of  lubricating  mixtures. 

ANALYSIS  OF  COMMERCIAL  SOAP. 

The  determinations  required  in  the  examination  of  a  commercial 
soap  depend  largely  upon  the  purpose  for  which  it  is  intended. 
The  scheme  outlined  below  is  adaptable  to  almost  all  cases  as  it 
can  be  easily  extended  to  include  additional  determinations  or 
shortened  by  omitting  such  steps  as  are  unnecessary  when  only  a 
partial  analysis  is  required. 

A  cake  of  soap  exposed  to  the  air  dries  rapidly  at  the  surface, 
forming  a  horny  layer  which  to  some  extent  prevents  the  evapor- 
ation of  water  from  the  interior.  In  sampling  such  a  soap  it  is 
important  to  remember  this  variation  in  water  content  of  different 
parts  of  the  same  cake.  If  the  sample  is  to  represent  the  indi- 
vidual cake  as  it  existed  at  the  time  of  commencing  the  analysis,  a 
number  of  sections  through  the  entire  cake  should  be  taken  either 
by  slicing  or  by  means  of  a  cork  borer  or  a  cheese  or  butter  sampler. 
Often  the  purpose  of  the  analysis  is  to  show  the  composition  of 
the  sample  as  originally  sold,  in  which  case  the  dried  surface  should 
be  rejected  and  the  sample  taken  from  the  center  of  the  cake.  The 
sample  for  analysis  should  be  reduced  quickly  to  fine  shavings  and 
kept  in  a  tightly  stoppered  bottle. 

DETAILS  OF  DETERMINATIONS  INDICATED  IN  THE  SCHEME. 

/.    Determination  of  Water. 

If  the  soap  is  very  hard  and  dry  it  may  be  reduced  to  fine 
shavings  and  dried  on  a  watch-glass,  heating  first  for  some  time  at 

1 80 


SOAPS   AND    LUBRICANTS. 
OUTLINE  SCHEME  OF  ANALYSIS.* 


181 


FIRST  PORTION.  Dry  2  to  5  grams  of  soap  (I.)f  and  introduce  into  Soxhlet  ex- 
tractor, either  in  a  thimble  or  supported  by  a  firm  plug  of  cotton,  and  extract  with 
petroleum  ether. 


Solution.     Evap-  |       Residue.      Allow  petroleum  ether  to  evaporate  and  exhaust 
orate  the  solvent;  dry    thoroughly  with  boiling  water. 

and  weigh  the  residue 

as  unsaponificd  fat  Solution.  Decompose  with  a  consider- I  Residue.  Dry 
and  unsaponifiable  \  able  excess  of  standard  sulphuric  (or  hy-  j  and  weigh  as  insolu- 
drochloric)  acid,  separate  aqueous  solu-  ble  matter. 


tion  from  fatty  layer.      (HI.) 


Solution.        Add         Fatty  Layer.  Dry 

methyl    orange    and  and   weigh  as  fatly 

titrate  for   total  al-  and      resin      acids, 

kali.    Test  for  chlor-  (V.) 
ides  (or  sulphates) , 
sugar,  and  glycerol. 


Ignite  and  weigh 
as  insoluble  mineral 
matter.  (X.) 


SECOND  PORTION.     Exhaust  2  to  5  grams  of  the  fresh  sample  with  carefully  neutral- 
ized alcohol.      (VI.) 


Solution.  Add 
phenolphthalein  and 
titrate  for  free  caus- 
tic alkali  or  free 
fatty  acid.  (VII.) 


Residue.     Dry   and 
with  boiling  water. 


weigh.     (VIII.)     Exhaust    thoroughly 


Solution.     Divide  into  aliquot  parts. 

Residzie.  May  be 
examined  instead  of 

Add        methyl         Test   for    (and  if 
orange    and    titrate    necessary  determine) 
for  alkali  carbonate    sulphate,       b  or  ate, 
(and  borate,  silicate,    silicate,  and  alumi- 
or  aluminate  if  pres-    note.      (IX.) 
ent).      (IX.) 

residue  from  first 
portion  or  used  for 
additional  tests  or 
determina  t  ions. 
(X.) 

40°  to  60°  and  then  finishing  at  105°  to  110°.  Very  few  soaps 
can  be  completely  freed  from  moisture  in  this  way  but  some  others 
may  be  sufficiently  dried  for  the  ether  extraction  while  the  mois- 
ture determination  is  made  on  a  separate  sample  as  follows :  Dis- 
solve about  two  grams  of  soap  in  the  minimum  quantity  of  hot 
strong  alcohol  and  evaporate  on  clean  dry  sand.  Finish  the  dryr 
ing  at  no0  with  frequent  stirring,  a  small  rod  having  been  weighed 
with  the  dish.  For  approximate  determination  of  moisture  Allen 
recommends  Smith's  method:  Heat  5  to  JO  grams  of  finely  di- 
vided soap  in  a  large  porcelain  crucible  on  a  sand  bath  over  a  small 
Bunsen  flame.  Stir  continually  with  a  small  glass  rod  (weighed 


*  Allen  :  Commercial  Organic  Analysis,  Vol.  II.,  Part  I. 
t  Numbers  in  parenthesis  refer  to  sections  which  follow. 


1 82  ORGANIC  ANALYSIS. 

with  the  crucible)  having  a  roughened  end  to  facilitate  breaking  any 
lumps  of  soap  which  may  be  formed.  Continue  heating  until 
there  is  no  more  evidence  of  water  being  expelled,  then  test  by 
removing  the  burner  and  placing  a  cold  glass  at  once  over  the 
crucible.  If  no  moisture  condenses  on  the  glass  the  soap  is  con- 
sidered dry.  With  practice  the  drying  can  be  finished  in  20  to  30 
minutes.  Any  burning  of  the  soap  is  readily  detected  by  the 
odor.  The  results  are  said  to  be  reliable  to  0.25  per  cent. 

//.     Petroleum  Ether  Extract. 

Transfer  the  thoroughly  dried  soap  to  a  paper  "  fat  extraction 
thimble,"  plug  tightly  with  fat-free  cotton  and  treat  in  a  Soxhlet 
extractor,  heated  on  a  safety  water  bath  or  electric  heater,  with 
petroleum  ether  of  nearly  constant  boiling  point.  Regulate  the 
heating  so  that  the  extract  fills  to  the  syphoning  point  in  ten  or 
fifteen  minutes  and  continue  the  extraction  for  four  to  six  hours. 
Disconnect  the  extraction  apparatus  (observing  that  no  free  flame 
is  near)  as  the  solvent  flows  through  the  syphon  to  the  flask ;  re- 
move the  thimble,  reconnect  the  apparatus,  and  recover  the  solvent 
by  placing  a  wide  short  test-tube  in  the  space  previously  occupied 
by  the  thimble,  or  by  allowing  the  solvent  to  collect  in  this  space 
and  removing  it  before  it  reaches  the  top  of  the  syphon.  Having 
expelled  nearly  all  of  the  petroleum  ether,  heat  the  flask  contain- 
ing the  extract  in  a  boiling  water  oven  to  constant  weight. 

The  petroleum  ether  extract  of  a  commercially  pure  soap  may 
contain  unsaponified  fat  (or  free  fatty  acids,  which  are  now  largely 
used  in  soapmaking)  as  well  as  the  "  unsaponifiable  matter  "  of  the 
original  soap  grease.  Hydrocarbons,  phenols,  and  other  sub- 
stances soluble  in  petroleum  ether  may  also  be  found  in  mixed  and 
"  medicated  "  soaps.  Allen  *  gives  directions  for  the  systematic 
examination  of  this  extract  including  the  quantitative  determination 
of  phenol  if  present. 

///.     Liberation  of  Fatty  and  Resin  Acids. 

Decompose  the  water  solution  of  the  soap  by  adding  a  consider- 
able excess  (allow  5  to  7  c.c.  of  normal  acid  for  each  gram  of  dry 
soap)  of  normal  or  half-normal  sulphuric,  nitric,  or  hydrochloric 
acid.  Add  the  entire  amount  of  acid  at  once,  boil,  stir  thor- 
oughly for  some  minutes,  and  keep  the  solution  hot  until  the  fatty 

*  Commercial  Organic  Analysis,  Vol.  II.,  Ft.  I.  (3d  Ed.),  pp. ^279-284. 


SOAPS   AND    LUBRICANTS.  183 

acids  collect  at  the  surface,  leaving  the  water  solution  nearly  clear, 
then  complete  the  separation  as  in  the  determination  of  insoluble 
acids  in  butter. 

It  is  convenient  to  liberate  the  fatty  acids  in  a  tared  beaker  in 
which  they  can  afterward  be  dried  and  weighed  (V.).  If  the  fatty 
acids  are  liquid  at  ordinary  temperature  or  form  a  cake  too  soft  to 
be  handled  conveniently,  a  known  weight  of  dry  bleached  beeswax 
or  stearic  acid  may  be  added  to  the  hot  solution.  The  fatty  acids 
become  incorporated  with  the  wax  and  on  cooling  a  firm  cake  is 
obtained. 

IV.     Solution  Separated  from  Fatty  Acids. 

This  solution  contains,  in  the  form  of  sulphate  (or  chloride  if 
hydrochloric  acid  be  used  to  decompose  the  soap),  all  the  alkali 
originally  present  as  soap,  as  carbonate  (silicate  or  borate),  or  as 
hydroxide.  On  titrating  this  solution  with  alkali,  using  methyl 
orange  as  indicator,  the  amount  of  acid  found  to  have  been  neu- 
tralized gives  a  measure  of  the  total  alkali  of  the  soap.  Unless 
potash  is  known  to  be  present,  this  total  alkali  is  usually  calcu- 
lated as  sodium  oxide.  The  solution  also  contains  any  chlorides 
or  other  soluble  salts,  soluble  fatty  acids,  glycerol,  sugar,  etc., 
which  the  soap  may  have  contained.  After  titration  the  solution 
can  be  diluted  to  a  known  volume  and  separate  portions  taken 
for  qualitative  tests  and  quantitative  determinations. 

When  sulphuric  acid  has  been  used  to  liberate  the  fatty  acids, 
chlorides  can  be  determined  in  a  portion  of  the  neutralized  solution. 
If  it  is  desired  to  determine  both  chloride  and  sulphate  in  this 
solution  the  soap  can  be  decomposed  by  means  of  standard  nitric 
acid.  It  is  usually  more  convenient  to  test  for  sulphates  in  the 
residue  from  the  alcohol  extraction  as  described  below. 

Soluble  fatty  acids  will  be  found  in  this  solution  if  the  liberated 
acids  of  cocoanut  or  palmnut  oil  soaps  are  washed  with  hot  water, 
as  is  often  recommended.  When  the  fatty  acids  are  separated 
cold  and  washed  with  cold  water  only,  the  amount  dissolved  can 
usually  be  neglected  without  appreciable  error. 

Sugar,  if  present,  is  detected  in  a  part  of  this  solution.  After 
further  treatment  with  acid  to  ensure  complete  hydrolysis,  the 
invert-sugar  is  determined  either  volumetrically  or  gravimetrically 
(Chapter  V.).  Sugar  may  also  be  determined  by  means  of  the 
polariscope  using  a  separate  portion  of  the  sample  and  precipi- 
tating the  fatty  acids  as  insoluble  barium  soaps.* 

*Freyer:    Oesterr.  Chem.  Ztg.,  1900,  3,  25  ;  Analyst,  1900,  25,  127. 


1 84  ORGANIC  ANALYSIS. 

In  the  absence  of  sugar  and  oth  er  interfering  substances,  glycerol 
can  be  determined  by  treating  a  portion  of  the  neutralized  solu- 
tion directly  with  sulphuric  acid  and  standard  dichromate  as  de- 
scribed in  Chapter  III.  (Hehner's  method).  Since  the  results 
thus  found  are  often  too  high,  because  of  the  presence  of  organic 
impurities,  Lewkowitsch  recommends  *  the  following  method : 
Decompose  the  water  solution  of  the  soap  with  sulphuric  acid, 
separate  the  fatty  acids,  neutralize  the  filtrate  with  barium  carbon- 
ate, evaporate  to  a  syrup,  and  extract  with  a  mixture  of  3  parts  95 
per  cent,  alcohol  and  I  part  ether.  The  glycerol  thus  obtained  can 
be  determined  by  the  acetin  method  after  complete  removal  of 
alcohol,  but  results  sufficiently  accurate  for  ordinary  work  can  be 
obtained  by  drying,  weighing,  and  burning  the  glycerol  as  in  the 
analysis  of  fermented  liquors.  The  method  for  glycerol  in  wines 
(Chapter  III.)  can  also  be  used  to  separate  glycerol  from  sugar  in 
analyzing  a  soap  containing  both  of  these. 

V.     Mixed  Fatty  and  Resin  Acids. 

The  mixture  of  acids  liberated  as  already  described  (III.)  is  dried 
to  constant  weight  in  a  boiling  water  oven  in  a  weighed  flat  bot- 
tomed dish  or  beaker  as  in  the  determination  of  the  insoluble  acids 
in  butter  fat.  The  weight  having  been  found,  test  a  portion  for 
resin  acid  by  the  Liebermann-Storch  reaction  as  described  under 
drying  oils  (Chapter  X.).  In  the  absence  of  resin  acids  it  may  be 
possible  to  show  the  nature  of  the  fat  from  which  the  soap  was 
made,  by  examining  the  mixed  fatty  acids  according  to  the  meth- 
ods used  in  identifying  fats  and  oils  as  described  in  Chapters  IX. 
and  X.,  consulting  special  works  such  as  those  of  Lewkowitsch  or 
Benedikt-Ulzer  for  the  "  constants  "  which  cannot  be  inferred  from 
the  properties  of  the  corresponding  fats.  The  separation  of  fatty 
and  resin  acids  is  best  accomplished  by  Twitchell's  method  based 
upon  the  difference  of  behavior  of  these  acids  when  exposed  in 
alcoholic  solution  to  the  action  of  hydrochloric  acid.  By  this 
treatment  fatty  acids  are  converted  to  ethyl  esters  while  resin  acids 
remain  practically  unchanged.  The  method  is  carried  out  by 
Lewkowitsch f  as  follows:  Weigh  2  to  3  grams  of  the  mixed 
acids  in  a  flask,  dissolve  in  10  times  their  volume  of  absolute  alco- 
hol, immerse  the  flask  in  cold  water,  and  pass  a  current  of  dry 

*Oils,  Fats,  and  Waxes  (3d  Ed.),  p.  io3i. 
fOils,  Fats,  and  Waxes  (3d  Ed.),  p.  394. 


SOAPS   AND    LUBRICANTS.  185 

hydrochloric  acid  gas  through  the  solution  for  an  hour;  then 
dilute  the  contents  of  the  flask  (which  will  have  separated  into  two 
layers)  with  5  times  its  volume  of  water  and  boil  until  the  aqueous 
solution  has  become  clear.  Transfer  the  contents  of  the  flask  to  a 
separating  funnel  by  means  of  50  cc.  of  petroleum  ether  (boiling 
below  80°) ;  shake,  allow  to  separate,  draw  off  the  acid  solution,  and 
wash  the  petroleum  ether  layer  once  with  water.  After  the  latter 
has  separated  completely  and  been  removed,  add  a  solution  of  0.5 
gram  of  potassium  hydroxide  and  5  c.c.  of  alcohol  in  50  c.c.  of 
water ;  shake,  and  allow  to  separate.  The  ethyl  esters  remain  dis- 
solved in  the  petroleum  ether  while  the  resin  acids  are  extracted 
by  the  dilute  alkaline  solution  forming  soaps.  Draw  off  the 
soap  solution,  wash  the  petroleum  ether  solution  again  with  dilute 
alkali,  unite  the  alkaline  solutions ;  liberate  the  resin  acids  by  means 
of  hydrochloric  acid,  collect,  dry,  and  weigh  them  as  in  the  deter- 
mination of  liberated  fatty  acids. 

The  resin  acids  can  be  titrated  after  washing  free  from  hydro- 
chloric acid,  instead  of  being  separated  and  weighed.  The  volu- 
metric method  is  more  rapid  than  the  gravimetric  but  necessitates 
the  assumption  of  a  combining  weight  (346)  for  the  resin  acids, 
which  is  liable  to  considerable  inaccuracy.  According  to  Lewko- 
witsch,  the  results  by  the  volumetric  method  are  likely  to  be  too 
high ;  those  by  the  gravimetric  method  too  low. 

VI.     Extraction  with  Alcohol. 

Dry  soap  can  be  extracted  with  95  per  cent,  alcohol ;  for  wet 
soap  stronger  alcohol  should  be  used  so  that  after  taking  up  the 
moisture  of  the  sample  it  will  still  be  too  strong  to  dissolve  an  appre- 
ciable amount  of  carbonate.  The  alcohol  to  be  used  must  first  be 
very  carefully  neutralized,  using  phenolphthalein  as  indicator.  In 
this  neutralization  there  is  danger  of  adding  an  excess  of  alkali 
unless  it  is  remembered  that  the  full  pink  color  of  the  indicator  will 
not  appear  in  alcohol  of  this  strength.  If  difficulty  is  experienced 
in  detecting  the  neutral  point  a  small  amount  of  the  alcohol  can  be 
removed  and  mixed  with  an  equal  volume  of  boiling  water  to  bring 
out  the  color  of  the  indicator. 

While  the  extraction  of  the  soap  with  alcohol  is  often  carried 
out  in  open  vessels,  filtering  and  washing  in  the  ordinary  way,  it  is 
usually  more  satisfactory  to  use  the  Soxhlet  extractor.  The  soap 
can  be  put  in  a  paper  thimble  as  in  the  petroleum  ether  extraction 


1 86  ORGANIC  ANALYSIS. 

or  between  plugs  of  cotton  in  a  glass  tube  with  perforated  bottom. 
In  the  latter  case  the  progress  of  the  extraction  can  be  watched 
without  disconnecting  the  apparatus.  When  the  extraction  is  com- 
plete the  residue  should  be  in  powder  form.  If  distinct  pieces 
remain  these  may  contain  soap  which  has  been  protected  from  the 
action  of  the  alcohol  by  the  formation  of  a  layer  of  insoluble  salts. 
In  this  case  remove  and  crush  the  residue,  replace,  and  extract 
again. 

VII.     Free  Caustic  Alkali  or  Fatty  Acid. 

To  the  alcoholic  extract  add  a  few  drops  of  neutralized  phe- 
nolphthalein  solution.  If  the  solution  reacts  alkaline,  titrate  with 
tenth-normal  acid  for  caustic  alkali ;  if  acid,  titrate  with  tenth- 
normal  alkali  for  free  acid.  For  a  further  discussion  of  this  extract 
including  a  rapid  method  for  the  partial  analysis  of  soaps,  see 
Allen,  /.  c.,  pp.  291-294. 

VIII.     Residue  Insoluble  in  Alcohol. 

It  is  advisable  to  dry  and  weigh  this  residue  so  that  the  percent- 
age of  impurities  not  actually  determined  can  be  found  by  differ- 
ence. A  microscopic  examination  may  also  be  of  use  in  deter- 
mining the  subsequent  treatment.  Starch  and  gelatine  if  present 
could  be  separated  from  carbonate,  borate,  and  sulphate  by  dis- 
solving the  latter  salts  in  cold  water;  but  silicate  would  probably 
be  incompletely  dissolved,  and  it  is  therefore  better  as  a  rule 
to  extract  with  hot  water  and  to  use  separate  portions  of  the  soap, 
if  necessary,  for  the  determination  of  starch  and  gelatin.  In  such 
a  case  extract  the  soap  with  alcohol  and  in  the  residue  deter- 
mine starch  as  described  in  Chapter  VII.,  or  determine  nitrogen 
by  the  Kjeldahl  method  and  calculate  the  corresponding  amount  of 
gelatin,  taking  the  nitrogen  content  of  the  latter  as  17.9  per  cent* 

IX.  Carbonate,  Silicate,  Borate,  and  Aluminate. 
Add  to  the  water  extract  from  the  residue  insoluble  in  alcohol 
an  excess  of  standard  acid  and  boil  to  ensure  decomposition  of 
the  silicate.  If  it  is  important  to  distinguish  quantitatively  be- 
tween carbonate  and  the  other  alkaline  salts  present,  the  carbonic 
acid  given  off  during  this  boiling  can  be  collected  and  weighed. 
Add  methyl  orange  as  indicator  and  titrate  with  standard  alkali  to 
determine  the  total  amount  of  alkali  which  was  present  as  carbon- 
ate, silicate,  borate,  and  aluminate. 

*  Richards  and  Gies  :  Amer.  Journ.  Physiol.,  1902,  7,  129. 


SOAPS   AXD    LUBRICANTS.  187 

To  a  portion  of  the  solution  add  hydrochloric  acid  in  excess, 
evaporate  to  small  volume  and  test  for  boric  acid  by  means  of  turmeric 
paper;  when  dry,  heat  at  110°,  take  up  with  dilute  hydrochloric 
acid,  filter  out  and  determine  silica,  if  present. 

Other  portions  of  the  solution  or  the  filtrate  from  silica  can  be 
used  for  the  detection  and  determination  of  sulphates,  aluminateSj 
etc. 

Jf.     Insoluble  Matter. 

This  residue  should  be  dried  to  constant  weight  at  100°,  a  por- 
tion examined  microscopically  and  the  remainder  ignited  and 
weighed.  If  over  one  per  cent,  of  insoluble  mineral  matter  is 
found  it  should  be  analyzed.  Among  the  substances  which  may 
be  found  in  this  residue  are  oatmeal,  bran,  sawdust,  clay,  chalk, 
steatite,  infusorial  earth,  pumice,  sand,  mineral  pigments,  etc. 

CALCULATION  AND  INTERPRETATION  OF  RESULTS. 

In  the  case  of  hard  soap,  the  results  of  the  partial  analysis 
usually  required  may  be  reported  as  follows : 

Water;  unsaponified  fat  and  unsaponifiable  matter;  fatty  and 
resin  anhydrides  (97  per  cent,  of  the  weight  of  free  acids) ;  sodium 
oxide  combined  as  soap;  sodium  hydroxide;  sodium  carbonate; 
insoluble  organic  matter;  insoluble  mineral  matter.  It  is  well  to 
report  also  the  total  alkali  in  terms  of  sodium  oxide. 

The  purpose  for  which  a  soap  is  intended  must  be  known  before 
an  opinion  as  to  its  quality  can  safely  be  formed.  In  most  cases 
the  percentage  of  alkali  combined  as  soap  is  the  best  measure  of 
the  amount  of  actual  soap  in  the  material,  but  for  special  purposes 
the  presence  or  absence  of  other  constituents  is  often  of  greater 
importance. 

Toilet  soaps  should  contain  as  little  free  alkali  (either  caustic  or 
carbonate)  as  possible.  Alder- Wright  divided  toilet  soaps  into 
three  classes  according  to  the  proportion  of  free  alkali  to  alkali 
combined  as  soap.  The  first  class  included  those  soaps  which 
contained  less  than  2.5  per  cent,  as  much  free  as  combined 
alkali  ;  the  second,  those  in  which  the  percentage  was  2.5  to  7.5  ; 
the  third,  those  containing  over  7.5  per  cent,  as  much  free  as  com- 
bined alkali.  In  judging  the  quality  of  toilet  soaps  it  is  also 
important  to  consider  the  proportions  and  nature  of  all  foreign 
matter,  the  amount  of  water,  the  hardness  of  the  soap  and  in  some 
cases  the  origin  must  be  sought  by  an  examination  of  the  fatty 


1 88  ORGANIC  ANALYSIS. 

acids.  The  more  expensive  •'  transparent "  toilet  soaps  may  con- 
tain alcohol  or  glycerine ;  in  cheaper  grades  a  similar  appearance 
is  obtained  by  the  addition  of  sugar. 

Household  soaps  are  made  from  cheaper  and  softer  fats  than 
those  used  for  toilet  soap.  Alkali  in  the  form  of  carbonate,  silicate, 
or  borate  is  not  objectionable  unless  present  in  excessive  amount. 
No  appreciable  amount  of  sugar  or  glycerol  is  likely  to  be  present. 
Scouring  soaps  often  contain  large  amounts  of  pulverized  quartz, 
infusorial  earth,  etc.,  and  are  sometimes  strongly  alkaline  with 
sodium  carbonate  or  hydroxide. 

The  qualities  of  soaps  for  special  arid  technical  purposes  are  dis- 
cussed in  several  of  the  following  works. 

REFERENCES. 

Alder- Wright :  Animal  and  Vegetable  Fixed  Oils,  Fats,  Butters, 
and  Waxes.  London,  1894.  (Revised  by  Mitchell,  1903.) 

Allen :  Commercial  Organic  Analysis,  Vol.  II.,  Part  I. 

Benedikt-Ulzer :  Analyse  der  Fette  und  Wachsarten. 

Devine  :  A  Method  of  Determining  Free  Alkali  in  Soaps,  Journ. 
Amer.  Chem.  Soc.t  1900,  22,  693.  The  Determination  of  Rosin 
in  Soaps,  Chem.  Eng.,  1905,  i,  207. 

Doane :  The  Disinfectant  Properties  of  Washing  Powders,  Bui- 
79,  Maryland  Agricultural  Experiment  Station,  1902. 

Freidrich :  Seifenanalysen ;  4  Bericht  des  Vereins  gegen  Ver- 
falschung  der  Lebensmittel,  etc.,  in  Chemnitz,  1902,  132;  Ztschr. 
Unters.  Nahr.  Genussm.,  1903,  6,  851. 

Heermann:  Ueber  die  Bestimmung  geringer  Mengen  Aetzna- 
tron  und  Soda  in  Seifen,  Chem.  Ztg.,.  1904,  28,  531. 

Heller:  Ueber  die  Bedeutung  von  Seifenzusatz  zu  Desinfections- 
mitteln,  Arch.  Hygiene,  1903,  47,  213. 

Henriques:  Specielle  Methoden  der  Oel-  und  Fettindustrie.  II. 
Seife,  Lunge's  Chemische-technische  Untersuchungsmethoden  (4th 
Ed.),  III.,  130. 

Henriques  and  Mayer:  Neue  Methode  zur  Bestimmung  des 
gesammt,  des  freien  und  des  kohlensauren  Alkali  in  Seifen,  Ztschr. 
angew.  Chem.,  1900,  785. 

Holde  and  Marcuson :  Die  quantitative  Bestimmung  von  Ko- 
lophonium  neben  Fettsauren  in  Seifen,  Fetten,  Ceresin,  u.  s.  w., 
Mitt.  Kgl.  techn.  Versuchstalt.  Berlin,  1902,  20,  40;  Ztschr.  Unters. 
Nahr.  Genus sm.,  1904,  7,  58. 


SOAPS   AND    LUBRICANTS.  189 

Hurst:  Practical  Manual  of  the  Manufacture  of  Soaps,  1898. 

Lewkowitsch  :  Chemical  Technology  and  Analysis  of  Oils,  Fats, 
and  Waxes. 

Pennsylvania  Railroad:  Specifications  for  Soap,   1895. 

Rodet :  Experiences  zur  la  Valeur  Antiseptique  du  savon  com- 
mun,  Revue  d*  Hygiene,  1905,  27,  301. 

Van  Slyke  and  Urner :  The  Composition  of  Commercial  Soaps 
in  Relation  to  Spraying,  Bui.  257,  N.  Y.  Agricultural  Experiment 
Station  (Geneva),  1904. 

EXAMINATION    OF    LUBRICATING   GREASES. 

Lubricating  greases  are  usually  mixtures  of  soaps  with  fats, 
hydrocarbons,  rosin,  or  tar,  containing  water  and  sometimes  large 
amounts  of  mineral  matter.  On  melting,  the  grease  often  separates 
into  a  soap  solution  and  an  oily  layer.  The  soaps  used  in  making 
such  lubricants  may  contain  salts  of  sodium,  calcium,  or  heavy 
metals  with  either  fatty  or  resin  acids.  Some  greases  consisting 
essentially  of  fats  and  hydrocarbons  melt  at  the  temperatures  to 
which  they  are  subjected  in  use  and  may  therefore  be  examined 
in  the  melted  state  by  the  methods  used  for  lubricating  oils.  For 
most  greases,  however,  it  is  necessary  to  adapt  the  analytical 
method  to  the  nature  of  the  mixture  to  be  examined  in  each  case, 
since  the  composition  of  these  greases  is  too  variable  to  allow  the 
use  of  any  fixed  system  of  examination.  It  may  often  be  neces- 
sary to  resort  to  a  combination  of  the  methods  used  in  the  analysis 
of  soaps,  fats,  and  lubricating  oils,  For  detailed  information  on 
the  composition  and  testing  of  lubricating  greases  the  reader  is  re- 
ferred to  the  reference  books  cited  at  the  end  of  this  chapter  and 
to  a  review  by  Conradson :  Journ.  Amer.  Chem.  Soc.,  1904,  26, 
705-712. 

EXAMINATION   OF    LUBRICATING   OILS. 

A  thorough  examination  of  lubricating  oil  involves :  (i)  the  de- 
termination of  the  nature  of  the  oil  and,  if  it  is  a  mixture,  the  pro- 
portion of  each  constituent ;  (2)  tests  to  determine  the  effici- 
ency and  safety  of  the  oil  as  a  lubricant  with  special  reference 
to  the  conditions  of  temperature,  pressure,  etc.,  to  which  it  will  be 
subjected  in  use.  Among  the  most  important  properties  of  lubri- 
cating oils  which  can  be  measured  in  the  laboratory  are  viscosity, 
acidity,  the  temperature  at  which  the  oil  solidifies,  and  the  flash- 
ing and  burning  points.  The  usefulness  of  other  determinations 
will  depend  upon  the  purposes  for  which  the  oil  is  intended. 


190  ORGANIC  ANALYSIS. 

DETERMINATION  OF  CONSTITUENTS. 

Pure  fatty  and  mineral  oils  are  largely  used  as  lubricants,  both 
singly  and  mixed  with  each  other  in  all  proportions.  Other  sub- 
stances are,  however,  often  added  to  reduce  the  cost  or  to  increase 
viscosity  of  the  oil,  among  the  most  common  being  rosin  oils  and 
"  gelatin  oils "  containing  aluminum  oleate  or  other  soaps.  A 
better  but  more  expensive  means  of  increasing  viscosity  is  to  use 
castor  oil  or  a  "  blown  "  oil. 

In  beginning  the  examination  of  a  lubricating  oil,  note  carefully 
any  color,  odor,  turbidity,  or  fluorescence  which  may  aid  in  identi- 
fying the  oil  or  detecting  foreign  substances  The  presence  of 
soap  is  easily  shown  by  igniting  a  portion  of  the  oil,  as  refined 
fatty  and  mineral  oils  should  not  yield  over  0.05  per  cent,  of  ash. 
Rosin  oil  can  be  detected  by  the  Liebermann-Storch  reaction. 

After  making  these  preliminary  observations  the  saponification 
number  should  be  determined  as  this  may  show  the  sample  to  be 
a  practically  pure  fatty  or  mineral  oil.  In  saponifying  mixtures 
consisting  largely  of  heavy  mineral  oil  there  is  difficulty  in  secur- 
ing sufficient  contact  between  the  sample  and  the  alcoholic  potash 
solution  even  though  petroleum  ether  or  gasoline  be  added.  In 
such  cases  a  Soxhlet  extractor  can  be  placed  between  the  flask  and 
the  reflux  condenser.*  The  intermittent  syphoning  of  the  con- 
densed solvent  from  the  extractor  into  the  saponification  flask 
mixes  the  contents  and  facilitates  saponification.  In  order  to 
diminish  the  volume  of  solvent  required  and  the  interval  between 
stirrings,  the  body  of  the  extractor  is  rilled  with  glass  beads. 
Having  found  the  saponification  number  (Chapter  IX.),  if  the 
sample  appears  to  be  a  mixture  of  fatty  and  mineral  or  rosin  oil 
the  proportions  of  saponifiable  and  unsaponifiable  matter  are  found 
either  by  separating  and  weighing  the  latter  or  by  estimating  the 
former  from  the  amount  of  fatty  acids  recovered  from  the  soap 
solution  after  saponification. 

Determination  of  Unsaponifiable  Oils. 

Weigh  2  to  10  grams  of  oil  (depending  upon  the  saponification 
number  and  the  method  to  be  followed),  saponify  by  heating 
with  alcoholic  potash  on  a  water  bath  ,f  evaporate  off  the  alcohol, 

*  Conradson  :  Journ.  Amer.  Chem.  Soc.,  1904,  26,  672. 

I  See  also  the  method  involving  cold  saponincation  given  by  Fahrion  :  Ztschr. 
angew.  Chem.,  1898,  267. 


SOAPS   AND    LUBRICANTS.  191 

and  separate  the  unsaponifiable  matter  by  one  of  the  following 
methods. 

Method  of  Immiscible  Solvents.  —  To  the  residue  from  the  evap- 
oration of  alcohol  add  75  c.c.  of  water,  stir  thoroughly,  transfer  to  a 
separatory  funnel,  add  about  an  equal  volume  of  petroleum  ether 
or  washed  ethyl  ether,  close  the  funnel,  shake  vigorously,  and  allow 
to  stand  over  night  or  until  the  aqueous  and  ethereal  solutions 
separate  completely.  Draw  off  the  aqueous  layer  into  another 
separatory  funnel ;  wash  it  again  with  ether  and  the  ethereal  layer 
again  with  water;  repeat  if  necessary.  Finally  unite  the  ether 
solutions  in  a  weighed  flask,  distil  off  the  ether,  and  dry  the  unsa- 
ponifiable oil  to  constant  weight  in  a  boiling  water  oven. 

If  desired,  the  fatty  acids  can  be  recovered  from  the  aqueous 
soap  solution  by  adding  an  excess  of  mineral  acid  and  shaking 
with  ether  or  by  separating  the  fatty  acids  as  in  soap  analysis. 

The  principal  objection  to  the  separation  by  immiscible  solvents 
is  that  emulsions  frequently  form  in  the  separating  funnel  which 
remain  even  on  standing  for  a  day  or  more.  The  addition  of  I  to 
2  c.c.  of  alcohol  often  helps  to  break  the  emulsion  but  if  more  alco- 
hol is  added  it  tends  to  carry  soap  into  the  ether  layer.  The  sep- 
aration of  the  solvents  is  also  facilitated  by  chilling  the  funnel  and 
twirling  it  gently  or,  if  the  apparatus  is  available,  by  whirling  in  a 
centrifuge.  Petroleum  ether  dissolves  less  soap  than  ethyl  ether 
and  gives  less  troublesome  emulsions  but  does  not  always  extract 
the  unsaponifiable  matter  completely. 

Extraction  of  the  Dry  Soap.  —  To  avoid  difficulties  of  the  preced- 
ing method  A.  C.  Wright  *  recommends  the  following :  Saponify 
10  grams  of  oil  using  5  grams  of  caustic  potash  ;  after  evaporating 
off  the  alcohol,  add  8  grams  of  sodium  bicarbonate  and  20  c.c.  of 
pure  methyl  alcohol,  stir  well  and  evaporate,  add  10  c.c.  more  of 
methyl  alcohol  and  25  grams  of  precipitated  chalk,  mix  well,  dry 
on  a  water  bath  and  then  for  a  few  minutes  at  1 10°.  Transfer  the 
thoroughly  dried  mixture  quickly  to  a  Soxhlet  extractor  and  ex- 
tract the  unsaponifiable  matter  with  petroleum  ether.  Dry  the 
extract  to  constant  weight  in  a  boiling  water  oven  and  weigh. 

The  mixture  of  calcium  carbonate  and  soap  from  which  the  un- 
saponifiable matter  has  been  extracted  can  be  treated  with  hydro- 
chloric acid  to  dissolve  the  carbonate  and  liberate  the  fatty  acids 
which  can  then  be  separated  and  examined  further. 

*  Analysis  of  Oils  and  Allied  Substances,  p.  III. 


I92  ORGANIC  ANALYSIS. 

Estimation  and  Identification  of  Fatty  Oils. 

From  the  weight  of  fatty  acid  recovered  as  described  above,  the 
percentage  of  fatty  oil  can  be  calculated  on  the  assumption  that  the 
oil  yields  95  per  cent,  of  insoluble  fatty  acids.  The  result  thus 
found  serves  as  a  check  upon  the  direct  determination  of  unsaponi- 
fiable  oil. 

If  only  fatty  and  mineral  oils  are  present  and  the  percentage  of 
the  former  is  small,  it  can  be  estimated  with  sufficient  accuracy  for 
most  purposes  from  the  saponification  number,  since  the  fatty  oils 
which  are  likely  to  be  present  in  mixed  lubricants  do  not  vary 
greatly  in  their  saponification  numbers.  See  table  at  end  of  Chap- 
ter IX.  If  the  fatty  oil  is  identified  the  average  number  for  that 
species  of  oil  should  be  used  in  estimating  the  percentage. 

If  the  lubricant  consists  entirely  of  fatty  oil  with  a  known  small 
amount  of  inert  unsaponifiable  matter,  the  usual  methods  for  the 
identification  of  fatty  oils  can  be  employed.  Otherwise  the  identi- 
fication is  based  upon  the  examination  of  the  separated  fatty  acids. 

VISCOSITY. 
Apparatus  and  Methods. 

The  viscosity  of  an  oil  can  be  determined  either  by  measuring 
the  resistance  which  it  offers  to  the  movement  of  a  submerged 
solid,  or  by  observing  the  rate  at  which  it  flows  through  an  aper- 
ture under  given  conditions  of  temperature  and  pressure.  Torsion 
viscosimeters  such  as  that  of  Doolittle  *  depend  upon  the  first  prin- 
ciple but  those  depending  upon  the  measurement  of  the  rate  of 
flow  are  much  more  generally  used.  Viscosimeters  of  this  kind 
are  made  in  a  great  variety  of  forms  for  descriptions  of  which  the 
reference  books  at  the  end  of  the  chapter  can  be  consulted. 
Among  the  viscosimeters  most  commonly  used  in  the  United 
States  are  those  of  Redwood,  Engler,  and  Saybolt. 

Redwood's  viscosimeter  consists  of  a  cylinder  about  4.7  cm.  in 
diameter  and  8.7  cm.  high,  having  in  the  center  of  the  bottom  a 
cup-shaped  agate  jet  which  can  be  closed  by  means  of  a  spherical 
plug.  Inside  the  cylinder  is  a  small  fixed  bracket  of  thick  bent 
wire  with  an  upturned  point  to  indicate  the  height  to  which  the 
oil  should  extend  at  the  beginning  of  the  test.  The  apparatus  is 
jacketed  and  provided  with  a  closed  side  tube  and  a  revolving 

*  Journ.  Amer.  Chem.  Soc.,  1893,  15,  174. 


SOAPS   AND    LUBRICANTS.  193 

stirrer  so  that  determinations  can  be  made  at  high  temperatures  if 
desired.  The  apparatus  is  intended  to  deliver  50  c.c.  of  water  at 
15.5°  in  25.5  seconds,  but  as  the  rate  of  flow  is  influenced  by  many 
conditions  it  must  be  determined  by  each  observer  for  his  own 
apparatus  and  method  of  working.  The  instrument  must  be  very 
carefully  cleaned  and  dried  before  and  after  using  as  any  dust  (or 
scratches  caused  by  careless  cleaning)  in  the  agate  jet  will  alter  the 
rate  of  flow. 

To  use  the  apparatus  at  room  temperature  place  it  on  a  level 
support,  insert  the  plug,  and  fill  with  the  liquid  to  be  tested  until 
the  surface  comes  exactly  to  the  upturned  point  already  mentioned. 
Place  beneath  the  outlet  a  narrow- necked  flask  graduated  at  50  c.c., 
open  the  jet  by  lifting  the  ball  valve,  and  observe  the  time  required 
for  50  c.c.  to  flow  into  the  receiving  flask.  A  stop  watch  is  recom- 
mended but  is  not  absolutely  essential.  Repeat  the  determination 
as  often  as  is  necessary  to  obtain  an  average  result  having  a  prob- 
able error  of  less  than  2  per  cent.  Whatever  method  is  adopted 
for  expressing  the  results,  *  the  report  should  always  show  the 
actual  time  of  flow  for  the  oil  and  for  water  and  the  temperature 
at  which  the  test  was  made. 

In  working  at  any  temperature  other  than  that  of  the  room,  fill 
the  outer  jacket  of  the  viscosimeter  with  water,  or  for  temperatures 
above  95°  with  mineral  oil.  This  is  first  brought  to  the  required 
temperature,  the  oil  to  be  tested  (previously  brought  to  the  same 
temperature)  is  then  introduced  and  the  receiving  flask  is  placed 
in  a  bath  of  liquid  at  the  temperature  at  which  the  test  is  to  be 
made.  At  high  temperatures  great  care  must  be  taken  in  heating 
and  stirring  as  a  variation  of  i°  or  less  will  sometimes  make  an 
appreciable  difference  in  the  time  of  flow. 

The  Redwood  viscosimeter  is  generally  regarded  as  standard  in 
Great  Britain  having  been  adopted  f  by  the  War  Department,  the 
Scottish  Mineral  Oil  Association,  and  the  principal  railway  com- 
panies. Engler's  viscosimeter  occupies  a  similar  position  in 
Germany  and  the  "normal  apparatus"  of  this  form  is  now  manu- 
factured under  the  joint  control  of  the  Charlottenburg  Mechanisch- 
technische  Versuchsanstalt  and  the  Karlsruhe  Chemisch-technische 
Versuchsanstalt.  For  description  and  illustration  of  this  apparatus 

*  Results  are  sometimes  compared  with  standard  rape  oil  requiring  535  seconds  at 
15.5°.  Other  methods  of  expressing  viscosity  are  given  by  different  writers  ;  see  refer- 
ences at  the  end  of  the  chapter. 

t  Lewkowitsch  :  Oils,  Fats,  and  Waxes  (3d  Ed.),  p.  199. 


194  ORGANIC  ANALYSIS. 

see  Lewkowitsch's  Oils,  Fats,  and  Waxes  (3d  Ed.),  pp.  202-204. 
Directions  for  use  are  furnished  with  each  apparatus.  For  a  de- 
scription of  Saybolt's  viscosimeter  which  is  made  in  three  forms 
for  testing  different  types  of  oils  consult  Gill's  Oil  Analysis  (3d 
Ed.),  pp.  24-28. 

Significance  of  Results. 

Since  the  object  of  lubricating  with  oil  is  to  separate  the  moving 
surfaces  by  a  fluid  layer,  it  is  important  that  the  oil  have  sufficient 
viscosity  or  "  body  "  to  stay  in  place  and  keep  the  moving  surfaces 
apart  under  the  maximum  pressure  to  which  they  will  be  sub- 
jected. The  greater  the  pressure  the  more  viscous  the  oil  should 
be,  but  any  viscosity  beyond  that  which  is  necessary  to  keep  the 
oil  in  place  means  an  increase  of  fluid  friction  and  consequent  loss 
of  power.  The  viscosity  of  the  oil  is  therefore  the  most  direct 
indication  of  its  suitability  as  a  lubricant  under  given  conditions. 
For  several  reasons,  however,  the  viscosity  alone  is  not  a  conclu- 
sive measure  of  the  lubricating  power.  The  adhesion  to  solid  sur- 
faces which  prevents  the  displacement  of  the  oil  by  pressure  is  not 
always  directly  proportional  to  the  true  viscosity  or  internal  fric- 
tion. Oils  vary  greatly  in  the  rate  of  change  of  viscosity  with 
increasing  temperature  and  pressure.  The  viscosity  as  measured 
by  the  rate  of  flow  depends  not  only  upon  the  internal  friction  of 
the  oil,  but  also  to  some  extent  upon  its  adhesion  to  the  sides  of 
the  outlet  and  upon  the  specific  gravity.  Hence  it  is  not  to  be 
assumed  that  any  two  oils  having  the  same  viscosity  can  be  used 
interchangeably  as  lubricants.  In  order  to  duplicate  an  oil  which 
has  been  found  satisfactory  in  use,  the  kind  of  oil,  the  specific 
gravity,  and  the  viscosity  shall  all  be  specified. 

Viscosity  is  especially  important  in  dealing  with  mineral  oils 
because  of  the  ease  with  which  they  can  be  varied  in  this  respect, 
while  any  particular  kind  of  fatty  oil  varies  only  within  compara- 
tively narrow  limits.  For  a  full  theoretical  discussion  of  viscosity 
and  lubrication  the  work  of  Archbutt  and  Deeley  should  be  con- 
sulted. The  reader  must  also  be  referred  to  this  and  other  books 
and  articles  given  below  for  discussion  of  the  many  practical  con- 
siderations affecting  the  selection  of  lubricating  oils. 

ACIDITY. 

Weigh  accurately  5  to  10  grams  of  oil  in  a  250  c.c.  Erlenmeyer 
flask,  add  50  c.c.  of  neutralized  85  per  cent,  alcohol  containing 


SOAPS   AND    LUBRICANTS.  195 

phenolphthalein  as  indicator  and  titrate  with  standard  sodium  or 
potassium  hydroxide  shaking  vigorously  after  each  addition  until 
a  permanent  pink  color  is  obtained.  It  is  often  necessary  to  allow 
the  flask  to  stand  after  shaking  until  the  oil  separates  from  the 
alcohol  solution  before  observing  the  color  of  the  latter.* 

To  test  for  free  mineral  acid,  shake  10-15  grams  of  oil  with 
100  c.c.  of  warm  water  in  a  separatory  funnel,  allow  to  separate, 
draw  off  the  water,  filter  through  wet  paper,  cool,  and  add  methyl 
orange.  If  mineral  acid  is  found,  shake  the  oil  remaining  in  the 
funnel  repeatedly  with  small  portions  of  hot  water  until  all  min- 
eral acid  is  extracted,  filter  as  before,  add  the  filtrate  to  the  first 
portion  containing  methyl  orange  and  titrate  very  carefully  with 
standard  alkali.  Concentrate  the  neutralized  solution,  test  quali- 
tatively to  determine  the  nature  of  the  acid  and  calculate  the  per- 
centage. If  the  identification  of  the  mineral  acid  is  prevented  by 
the  presence  of  salts,  calculate  the  mineral  acidity  as  due  to  sul- 
phuric acid.  The  acidity  due  to  organic  acids,  or  the  total  acidity 
if  only  this  is  determined,  is  usually  calculated  as  percentage  of 
oleic  acid.  As  much  as  15  percent,  of  free  oleic  acid  is  some- 
times allowed  in  lubricating  oils.  The  best  grades  of  lard  oil  do 
not  contain  over  1.5  per  cent.  Free  mineral  acids  should  be 
absent. 

COLD  TEST  AND  CHILLING  POINT. 

The  "  cold  test  "  indicates  the  temperature  at  which  the  sample 
is  just  sufficiently  melted  to  flow;  the  "chilling  point"  that  at 
which  the  oil  begins  to  become  turbid  or  to  show  flocks  or  scales 
of  solid.  In  either  case  the  temperature  required  will  be  influenced 
by  details  of  manipulation,  so  that  an  arbitrary  method  must 
be  followed  to  obtain  strictly  comparable  results.  The  follow- 
ing directions  follow  the  procedure  of  the  Pennsylvania  railroad 
laboratory  .f 

Cold  Test.  —  Pour  about  25  c.c.  of  oil  into  an  ordinary  sample 
bottle  of  about  100  c.c.  capacity  and  introduce  a  short  stout  ther- 
mometer. Place  the  bottle  in  a  freezing  mixture  until  the  sample 

*  For  determining  acidity  in  very  dark  colored  fats  the  use  as  indicator  of  10  c.c. 
of  a  2  per  cent,  solution  of  "  Alkali  Blue  II  OLA  "  (Meister,  Lucius,  and  Brunig)  in 
99  per  cent,  alcohol  has  been  recommended  by  Freundlich  :  Oesterr.  Chem.  Ztg.,  1901, 
4,  441  ;  Ztschr.  Unter.  Nahr.-Genussm.,  1902,  5,  460. 

|  Motive  Power  Department  Specifications,  No.  75,  Altoona,  Pa.,  1895.  For  the 
method  and  apparatus  of  the  Chicago,  Burlington,  and  Quincy  Railroad  laboratory  see 
Stillman's  Engineering  Chemistry,  2d  Ed.,  pp.  352-354. 


196  ORGANIC   ANALYSIS. 

is  solid  throughout.  Remove  the  bottle  and  allow  the  solidified 
oil  to  soften,  stirring  and  mixing  by  means  of  the  thermometer 
until  the  mass  will  flow  from  one  end  of  the  bottle  to  the  other. 
The  reading  of  the  thermometer  at  this  point  is  the  "  cold  test." 

Chilling-  Point.  —  Usually  it  is  only  necessary  to  know  whether 
the  oil  remains  clear  for  a  given  number  of  minutes  at  a  given 
temperature.  Use  the  same  bottle,  amount  of  sample,  and  ther- 
mometer as  for  the  cold  test.  Expose  the  liquid  to  cold,  stirring 
with  the  thermometer  and  hold  at  the  required  temperature  for  the 
specified  time  (usually  ten  minutes).  If  the  oil  remains  transparent 
and  fre'e  from  flocks  or  scales,  it  meets  the  requirement  as  to 
chilling  test. 

If  it  is  required  to  find  the  chilling  point  the  procedure  is  similar, 
but  the  liquid  after  remaining  clear  as  described  is  exposed  to  a 
temperature  3°  lower,  allowed  to  stand  with  constant  watching 
and  occasional  stirring  with  the  thermometer  until  the  oil  is  as 
cold  as  the  bath,  repeat  this  cooling  until  opacity  or  flocks  or 
scales  begin  to  show.  The  reading  of  the  thermometer  when  this 
occurs  shows  the  "  chilling  point." 

For  further  information  on  the  cold  test  or  setting  point  see  the 
works  of  Archbutt  and  Deeley,  Gill,  Holde,  Lewkowitsch,  and 
Stillman. 

FLASHING  AND  BURNING  POINTS. 

For  the  most  accurate  results  closed  testers  such  as  are  used  in 
examining  illuminating  oils  should  be  employed,  but  as  such 
accuracy  is  not  required  in  testing  lubricants  it  will  usually  be 
sufficient  to  carry  out  the  test  without  the  use  of  any  special 
apparatus  as  follows :  * 

On  a  sand  bath  and  partly  imbedded  in  it  place  a  porcelain  dish 
about  6.5  cm.  in  diameter  and  2.5  cm.  deep.  Fill  the  dish  to 
about  0.6  cm.  of  the  top  with  the  oil  to  be  tested  and  hang 
a  thermometer  so  that  the  bulb  is  immersed  in  the  oil  at  the 
center  of  the  dish  without  touching  the  bottom.  Heat  by  means 
of  a  Bun?en  burner  and  as  the  flashing  point  is  approached 
test  at  each  third  or  fourth  degree  by  passing  the  test  flame  slowly 
entirely  across  the  dish  about  I  cm.  above  the  level  of  the  oil  and 
directly  in  front  of  the  thermometer.  Record  the  temperature  at 
which  the  first  flash  is  seen  as  the  flashing  point.  Continue  heat- 

*  Specifications  of  the  Pennsylvania  Railroad  Motive  Power  Department,  Altoona, 
Pa.,  1902. 


SOAPS   AXD    LUBRICANTS.  197 

ing  and  testing  in  the  same  way  until  the  liquid  takes  fire;  note 
the  temperature  as  the  burning  point.  Remove  the  thermometer 
and  blow  out  the  flame  or  smother  it  by  sliding  a  watch  glass  over 
the  dish. 

The  test  must  be  made  in  a  place  protected  from  drafts.  The 
test  flame  should  be  about  0.5  cm.  long  and  is  conveniently  ob- 
tained from  a  narrow  glass  jet  connected  with  the  ordinary  gas 
tubing.  The  flow  of  gas  is  easily  regulated  to  give  a  flame  of  the 
required  size.  The  heating  of  the  sandbath  should  be  so  regulated 
that  the  temperature  of  the  oil  rises  6°  to  8°  per  minute,  the  rise 
being  more  rapid  the  higher  the  flashing  point.  The  test  flame 
can  then  be  applied  every  half  minute. 

Any  variation  from  the  conditions  given,  either  in  the  size  and 
form  of  dish,  the  rate  of  heating  and  testing,  or  the  manner  of 
applying  the  test  flame  may  cause  an  appreciable  discrepancy  in 

the  result. 

ADDITIONAL  DETERMINATIONS. 

Additional  tests  and  determinations  are  frequently  required  to 
show  the  suitability  of  the  lubricant  for  the  particular  use  intended. 
friction  tests  on  oil  testing  machines  especially  designed  for  the 
work  are  sometimes  of  great  importance.  A  full  discussion  of  such 
mechanical  methods  of  testing  will  be  found  in  Archbutt  and 
Deeley's  Lubrication  and  Lubricants. 

Loss  by  evaporation  and  tendency  to  "gum  "  are  tested  by  heating 
a  small  amount  of  oil  on  a  watch  glass  for  several  hours  at  the 
highest  temperature  to  which  it  is  likely  to  be  subjected  in  use. 
The  oil  must  not  become  sticky  and  the  loss  of  weight  should 
usually  be  less  than  one  per  cent.  Suspended  matter  which  may 
be  invisible  in  a  dark  oil  is  detected  by  diluting  the  sample  with 
gasoline  or  petroleum  ether.  Antiflourescents  added  to  destroy  the 
fluorescence  or  "bloom  "  of  mineral  oils  can  often  be  detected  by 
boiling  I  c  c.  of  the  oil  with  3  c.c.  of  a  10  per  cent,  solution  of 
potassium  hydroxide  in  alcohol.  A  red  color  indicates  nitro- 
naphthalene  or  nitro- benzene,  which  are  the  principal  antifluores- 
cents  used.  According  to  Holde*  asphalt  pitch  is  used  to  thicken 
cylinder  oil  and  can  be  approximately  determined  as  follows:  Dis- 
solve 5  grams  in  125  c.c.  of  ether  at  15°,  add,  drop  by  drop  with 
constant  shaking,  62.5  c.c.  of  96  per  cent,  alcohol;  after  standing  5 

*Mitt,  KgL  Techn.  Versuchsanstalt,  Berlin,  1902,  20,  253;  Ztschr.  Unters.  Nahr.- 
Genussm.,  1903,  6,  855. 


I98  ORGANIC   ANALYSIS. 

hours  at  15°,  filter,  wash  with  a  mixture  of  alcohol  and  ether  (1:2 
by  volume)  until  nothing  more  than  a  trace  of  pitch-like  substance 
is  removed.  Dissolve  the  residue  in  benzol,  evaporate,  dry  one 
half  hour  at  105°,  and  weigh. 

For  other  tests  and  determinations  and  fuller  discussions  of  most 
of  those  here  given  the  reader  is  referred  to  the  works  given  for 
reference  below. 

REFERENCES. 

Alder- Wright :  Oils,  Fats,  Butters,  and  Waxes. 

Allen:  Commercial  Organic  Analysis,  Vol.  II.,  Part  II. 

Archbutt  and  Deeley:  Lubrication  and  Lubricants,  London, 
1900. 

Brannt :  Petroleum  and  its  Products,  Phila.,  1895. 

Gill:  Short  Hand-book  of  Oil  Analysis  (3d  Ed.),  Phila.,  1903. 

Holde:  Untersuchung  der  Schmiermittel,  Berlin,  1897. 

Lewkowitsch  :  Oils,  Fats,  and  Waxes  (3d  Ed.),  New  York,  1904. 

Stillman:  Engineering  Chemistry  (2d  Ed.),  Easton,  Pa.,  1900. 

Thurston :  Friction  and  Last  Work  in  Machinery  and  Millwork, 
New  York,  1885. 

IRedwood :  On  Viscosimetry,y"<?&r«.  Soc.  Chem.  Ind.,  1886,  121. 

lEger:  Grundsatze  fur  die  Priifung  von  Mineralschmierolen, 
Ztschr.  angew.  Chem.,  1904,  1577. 

Richardson  and  Hanson :  The  Valuation  of  Lubricants  with 
Special  Reference  to  Cylinder  Oi\s,Journ.  Soc.  Chem.  Ind.,  1905, 
24,315. 


CHAPTER  XIII. 
Proteids  and  Cereals. 

PROTEIDS   AND    RELATED    COMPOUNDS. 

The  proteids  are  very  complex  and  usually  amorphous  com- 
pounds, differing  in  composition  and  properties,  but  all  of  high 
molecular  weight  and  unknown  chemical  structure.  They  all  con- 
tain carbon,  hydrogen,  nitrogen,  sulphur,  and  oxygen  ;  some  also 
contain  phosphorous  and  a  comparatively  small  number  iron. 
The  limits  of  ultimate  composition  of  the  well-known  simple  pro- 
teids are  as  follows  :  * 

Carbon.  Hydrogen.  Nitrogen.  Sulphur.  Oxygen. 

$1-3-55'°          6-7-7-3          I5-5-I9-3          0.3-2.2          20.8-23.5  percent. 

Proteids  differ  in  their  solubilities  in  water,  salt  solutions,  and 
alcohol  but  are  all  insoluble  in  ether,  chloroform,  carbon  tetra- 
chloride,  carbon  bisulphide,  benzene,  and  petroleum  ether. 

The  proteid  compounds  are  of  the  greatest  importance  in  physi- 
ology and  food  chemistry,  being  the  fundamental  organic  con- 
stituents of  the  living  substance  of  both  animal  and  vegetable 
cells,  and  the  only  group  of  organic  compounds  which  is  strictly 
essential  to  the  nutrition  of  all  animals.  The  proteids  and  closely 
related  compounds  have  therefore  been  studied  and  classified 
largely  from  the  physiological  standpoint.  While  differences  exist 
both  in  classification  and  terminology,  the  following  is  believed  to 
represent  the  present  usage  of  the  majority  of  English-speaking 
physiologists  and  physiological  chemists.f 

SIMPLE  PROTEIDS. 

Albumins.  —  Soluble  in  water  and  dilute  salt  solutions;  precipi- 
tated by  adding  sufficient  alcohol  or  by  saturating  the  aqueous 
solution  with  ammonium  sulphate.  The  water  solutions  are 

*  Ultimate  analyses  of  a  number  of  proteids  have  recently  been  compiled  and  dis- 
cussed by  Osborne  :  Journ.  Amer.  Chem.  Soc.,  1902,  24,  160. 

f  Compare  American  Text-book  of  Physiology  and  reference  books  given  at  the  end 
of  the  chapter. 

199 


200  ORGANIC   ANALYSIS, 

coagulated    by    heating,    usually   at    7O°-73°    (Halliburton),   but 
sometimes  much  lower,  leucosin  coagulating  at  52°  (Osborne). 

Globulins.  —  Insoluble  in  pure  water  but  soluble  in  dilute  salt 
solutions.  Precipitated  by  alcohol  or  by  saturation  with  ammonium 
or  magnesium  sulphate ;  also  precipitated  from  salt  solutions  on 
removing  the  salt  by  dialysis.  Some  of  the  vegetable  globulins 
are  distinctly  crystalline  and  not  coagulable  by  heating. 

Alcohol  Soluble  Proteids.*  —  These  occur  especially  in  the  cereal 
grains.  Gliadin,  insoluble  in  water,  dilute  salt  solution,  or  absolute 
alcohol,  but  soluble  in  75  per  cent,  alcohol  is  the  best-known 
member  of  this  group. 

Alb uniinate s.  —  By  the  action  of  acid  or  alkali  upon  native  or 
coagulated  proteid  the  latter  is  converted  into  an  acid-  or  an  alkali- 
albumin  or  albuminate.  Both  acid  and  alkali  albuminates  are 
nearly  insoluble  in  water  and  dilute  salt  solution,  but  usually  dis- 
solve readily  in  water  containing  a  very  small  amount  of  acid  or 
alkali  and  are  precipitated  from  such  solutions  by  neutralization  at 
ordinary  temperature.  The  albuminates  are  precipitated  from 
nearly  neutral  solutions  by  the  general  precipitants  for  soluble 
proteids  (see  below) ;  but  a  very  minute  amount  of  acid  or  alkali 
is  sufficient  to  prevent  the  coagulation  of  albuminate  by  boiling. 
Acid  albumin,  also  called  syntonin,  is  formed  by  the  acid  ot  gastric 
juice  as  the  first  step  toward  peptic  digestion. 

Proteoses  and  Peptones.  —  These  are  products  derived  from  other 
proteids  by  digestion  or  by  simple  hydrolysis.  They  are  soluble 
in  water  and  not  coagulated  by  boiling  their  aqueous  solutions. 
No  sharp  line  can  be  drawn  either  between  proteoses  and  peptones 
or  between  peptones  and  the  simpler  nitrogen  compounds  which 
result  from  prolonged  digestion.  As  the  terms  are  generally  used, 
peptones  may  be  considered  the  final  products  of  digestion  or 
hydrolysis  which  are  still  proteids  as  judged  by  the  biuret  reaction 
(see  below)  and  are  precipitable  by  tannin  (perhaps  not  always  com- 
pletely) or  by  addition  of  strong  alcohol.  Proteoses  (albumoses)  are 
intermediate  products  between  albuminates  and  peptones.  In  ad- 
dition to  the  proteid  reactions  shown  by  peptones  the  proteoses  are 
precipitated  from  aqueous  solutions  at  ordinary  temperatures  by 
adding  acetic  acid  and  potassium  ferrocyanide  or  by  saturating  the 
solution  with  zinc  or  ammonium  sulphate. 

*  The  classifications  commonly  given  relating  especially  to  animal  proteids  omit  this 
group. 


PROTEIDS   AND    CEREALS.  201 

The  term  peptone  was  formerly  applied  to  all  digestion  products 
not  coagulated  by  boiling  and  is  still  popularly  used  in  the  same 
sense,  the  best  commercial  "  peptones  "  consist  largely  of  proteoses. 

Coagulated  Proteids. — This  group  includes  proteids  which  have 
been  coagulated  by  heating  or  by  the  action  of  reagents  or  enzymes. 
The  nature  of  the  changes  which  take  place  in  coagulation  is  not 
known.  The  coagulated  proteids  are  insoluble  in  water,  alcohol, 
salt  solutions,  or  very  dilute  acids.  They  are  dissolved  and  con- 
verted into  albuminates  by  acids  and  alkalies,  especially  on  heating. 

Some  plant  proteids  such  as  the  glutenin  of  wheat  are  insolu- 
ble in  the  neutral  solvents  but  dissolve  readily  in  very  dilute  alkali. 
These  may  be  native  insoluble  proteids  or  they  may  have  been 
coagulated  by  an  enzyme  in  the  plant. 

COMBINED  PROTEIDS. 

CJiromo -proteids.  —  There  are  compounds  of  simple  proteids  with 
coloring  matter  usually  containing  iron.  Haemoglobin  and  its 
derivatives  are  the  best-known  examples. 

Gly co  proteids.  —  Glyco-  or  gluco-proteids  are  compounds  of 
proteids  with  carbohydrates.  They  are  practically  insoluble  in 
water  but  easily  soluble  in  very  weak  alkalies.  On  boiling  with 
dilute  mineral  acids  they  yield  considerable  amounts  of  reducing 
sugars.  Mucins  and  mucoids  belong  to  this  group. 

Nucleo-proteids .  —  These  are  compounds  of  proteids  and  nuclein 
or  nucleic  acid,  an  organic  acid  yielding  phosphoric  acid  on  decom- 
position. True  nuclein  also  yields  on  decomposition  one  of  the 
purin  bases  and  is  thus  distinguished  from  paranuclein  which  it 
resembles  in  other  respects.  Nucleo-proteids  are  found  especially 
in  cell  nuclei  and  are  therefore  particularly  abundant  in  the  highly 
nucleated  cells  of  secreting  organs  such  as  the  liver,  pancreas,  etc. 

While  all  of  the  simple  proteids  are  laevorotatory,  the  nucleo- 
proteids  thus  far  studied  *  have  shown  dextro-rotation  and  the 
nucleic  acid  of  the  wheat  embryo  has  been  found  to  be  strongly 
dextrorotatory.! 

Paranucleo-proteids  or  Nude o~ albumins.  —  These  compounds  of 
proteids  with  paranuclein  are  often  classified  as  a  sub-group  of  the 
true  nucleo-proteids  which  they  resemble  closely  in  composition. 
Recently,  however,  the  purin  bases  have  been  extensively  studied 

*Gamgee  and  Jones:   Amer.  Journ.  Physiol.,    1903,  8,  447. 
tOsborne  :    Ibid.,  1003,  9,  69. 


* 


*/7> 


202  ORGANIC   ANALYSIS. 

and  shown  to  be  of  much  physiological  importance,  and  since  para- 
nucleins  do  not  yield  purin  bodies  on  decomposition,  the  paranucleo- 
proteids  are  now  frequently  classified  as  an  independent  group 
under  the  name  of  nucleo-albumins  and  are  sometimes  considered 
as  simple  proteids  containing  phosphorus.  Casein  and  vitellin 
(the  phosphorus-containing  proteid  of  egg  yolk)  are  examples  of 
this  group. 

ALBUMINOIDS  (ALBUMOIDS,  PROTEOIDS,  GELATINOIDS). 

This  division  includes  several  groups  of  compounds  differing 
considerably  from  each  other  but  all  closely  related  to  the  true  pro- 
teids included  in  the  above  groups  and  all  characterized  by  their 
resistance  to  reagents.  They  are  apparently  formed  from  proteids 
in  the  body  but  cannot  take  the  place  of  proteids  in  nutrition. 
Among  the  more  important  of  the  albuminoids  are  collagens  which 
yield  gelatins  on  boiling  with  water;  keratins  of  skin,  horn,  hair, 
feathers,  nails,  etc. ;  elastin  of  connective  tissue  ;  skeletins,  the  nitrog- 
enous compounds  of  the  skeletal  and  related  tissues  of  inverte- 
brates, including  the  characteristic  compounds  of  sponges  and 
silk. 

Gelatins  (and  collagens)  are  the  only  albuminoids  which  are 
likely  to  be  met  in  connection  with  the  true  proteids  in  foods,  etc., 
so  that  the  other  substances  mentioned  need  not  be  considered  in 
connection  with  the  terminology  and  reactions  given  below. 

OTHER  NITROGEN  COMPOUNDS  —  TERMINOLOGY. 
Among  the  organic  nitrogen  compounds  which  occur  with  pro- 
teids in  many  animal  or  vegetable  substances  are  the  lecithins  and 
related  compounds,  alkaloids,  and  the  so-called  "nitrogenous  ex- 
tractives" including  amines,  amids,  and  amino-acids.  Ammo- 
nium salts  and  nitrates  may  occur  in  plant  and  animal  tissues  but 
usually  only  in  minute  amounts.  They  may,  however,  be  added 
to  food  materials  as  preservatives.  In  most  natural  food  products 
the  total  amount  of  these  simpler  nitrogen  compounds  is  small  as 
compared  with  that  of  proteids  and  it  has  become  customary  in 
food  analysis  to  take  the  total  nitrogen  as  a  measure  of  the  proteids 
present,  so  that  the  percentage  of  proteids  as  reported  ordinarily 
means  the  percentage  of  nitrogen  multiplied  by  6.25,  this  factor 
being  based  on  the  assumption  that  proteids  contain  approximately 
16  per  cent,  of  nitrogen. 


PROTEIDS   AND    CEREALS.  203 

Protein  is  the  term  now  commonly  used  to  designate  the  results 
thus  arbitrarily  estimated  from  the  nitrogen  content,  proteids  being 
preferably  reserved  as  a  general  name  to  cover  the  simple  and 
combined  proteids  (and  sometimes  also  gelatin)  while  the  simpler 
nitrogen  compounds  are  grouped  together  as  non-proteids.  Beyond 
this  division  into  proteids  and  non-proteids  the  terminology  of  the 
nitrogen  compounds  of  foods  is  in  a  very  confused  state.  That 
recommended  by  the  Association  of  American  Agricultural  Col- 
leges and  Experiment  Stations  and  largely  used  by  agricultural 
writers  in  this  country  is  as  follows  :* 


[Albumins 
<  Globulins 
and  allies 


C  Simple  •{  Globulins 


Albuminoids  •< 

Derived 


Compound 

Collagens  or  gelatinoids 
(_  Non-proteids  :  Extractives  ;    amids,  amino  acids,  etc. 

The  most  important  point  to  be  noted  here  is  the  use  of  the 
word  "  albuminoids."  Formerly  this  was  used  synonymously 
with  proteids  to  designate  (i)  simple  and  combined  proteids  and 
(2)  gelatin  and  related  compounds.  As  the  differences  between 
these  two  classes  of  compounds  have  become  better  known  the 
majority  of  physiologists  and  physiological  chemists  have  confined 
the  term  albuminoids  to  the  latter  class  in  order  to  distinguish 
these  compounds  from  the  true  proteids,  while  in  the  agricultural 
terminology  just  quoted  the  compounds  of  the  first  class  are 
called  albuminoids.  Some  writers  propose  to  discard  the  term 
"  albuminoids  "  entirely,  retaining  "  simple  and  combined  pro  teids  ' 
and  using  for  the  related  compounds  some  new  term  such  as 
"  proteoids  "  or  "  albumoids." 

GENERAL  REACTIONS  OF  PROTEIDS. 

Color  Reactions. 

Among  the  most  important  color  reactions  are  :  — 
I  .   The  xanthoproteic  reaction.  —  Proteids  are  colored  yellow  by 
nitric  acid  of  1.2  specific  gravity  or  stronger.     The  color  is  in- 
tensified by  heating  and  changes  to  orange  or  red  on  treatment 

*U.    S.    Dept.    Agriculture,    Office   of  Experiment    Stations,    Bull.    65,  p.    117 
Armsby's  Principles  of  Animal  Nutrition,  p.  7 


204  ORGANIC   ANALYSIS. 

with  an  excess  of  ammonia.     According  to  Halliburton  *  this  re- 
action depends  upon  the  presence  of  an  oxybenzene  nucleus. 

2.  Mil/on  s  Reaction.  —  On    heating  with  Millon's  reagent f  (a. 
solution  of  mercuric  nitrate  containing  nitrous  acid)proteid  matter 
(including   gelatin)  gives  a  brick-red  coloration  which  according 
to  Nasse  is  also  due  to  the  presence  of  any  oxybenzene  nucleus  in 
the  proteid  molecule.     For  detailed   discussions  of  this  reaction 
the  papers  of  Vaubel  \  and  Nasse  §  should  be  consulted. 

3.  Adamkiewicz*  or  Tryptophan  reaction. — When  proteid  is  treated 
first  with  glacial  acetic  acid  and  then  with  concentrated  sulphuric 
a  violet  color  usually  appears.     Hopkins  and  Cole  ||  found  that  the 
reaction  occurred  only  when  the  acetic  acid  contained  traces  of 
glyoxylic  acid  and  that  a  better  reaction  is  obtained  by  mixing  the 
proteid  with  a  little  glyoxylic  acid  solution^  and  afterward  adding 
concentrated  sulphuric  acid.     The  color  then  appears  at  the  line  of 
contact  of  the  liquids.     Gelatin  does  not  give  the  reaction  (Ham- 
marsten).     According  to  Osborne  and  Harris  **  the  vegetable  pro- 
teids  vary  greatly  in  their  reactions,  some  of  them  giving  little  if 
any  violet  color.     This  reaction  is  due  to  the  presence  of  the  tryp- 
tophan  group  in  the  proteid  molecule,  tryptophan  being  probably 
skatol-amido-acetic  acid  (Hopkins  and  Cole). 

For  other  color  reactions  due  to  tryptophan  see  Cole :  Journ. 
Physiol.,  1903,30,  311. 

4.  Piotrowski  or  Biuret  Reaction. — On  adding  a  very  dilute  solu- 
tion of  copper  sulphate  drop  by  drop  to  a  proteid  solution  strongly 
alkaline  with  sodium  or  potassium  hydroxide  a  rose-red  to  violet 
•coloration  appears.     A  more  pronounced  rose  color  is  obtained 
with  proteoses  and  peptones  than  with  other  proteids.     To  test  for 

*  Schafer's  Textbook  of  Physiology,  I.,  47. 

f  To  prepare  the  reagent  dissolve  mercury  in  twice  its  weight  of  nitric  acid,  1.42  sp. 
gr. ,  and  dilute  the  solution  obtained  with  three  times  its  volume  of  water.  According 
to  Nasse  a  better  method  is  to  use  a  solution  of  mercuric  acetate  containing  a  few  milli- 
grams of  sodium  or  potassium  nitrite,  the  solution  having  been  recently  acidulated  with 
.acetic  acid. 

\Ztschr.  angew.  Chem.,  1900,  1125. 

^Arch.ges.  Physiol.  (P/luger),  1901,  83,  361. 

IJI  Proc.  Royal  Soc.,  1901,  68,  21  ;  Journ.  Physiol.,  27,  418  ;  29,  451. 

^[  Prepared  as  follows  :  Place  a  saturated  solution  of  oxalic  acid  in  a  tall  cylinder, 
add  lumps  of  sodium  amalgam  (about  60  grams  per  liter  of  solution),  allow  to  stand  as 
long  as  hydrogen  is  evolved  then  filter  and  dilute  the  solution  with  twice  its  volume  of 
water. 

** Journ.  Amer.  Chem.  Soc.,  1903,  25,  853. 


PROTEIDS   AND    CEREALS.  205 

peptones  in  a  solution  containing  other  proteids,  *  precipitate  the 
latter  by  saturation  with  zinc  sulphate,  filler,  to  the  filtrate  add 
caustic  soda  until  the  zinc  hydroxide  first  precipitated  is  completely 
dissolved,  then  add  a  few  drops  of  a  one  per  cent,  solution  of  cop- 
per sulphate.  This  test  is  said  to  show  as  little  as  I  part  of  pep- 
tone in  100,000  of  solution.  For  other  proteids  it  is  less  delicate. 
The  reaction  is  given  by  many  substances  other  than  proteids ; 
according  to  Scruff "f"  by  any  compound  containing  two  amino 
groups  united  either  directly  by  their  carbon  atoms  or  by  means 
of  a  third  carbon  or  a  nitrogen  atom.  Examples  of  these  three 
classes  of  compounds  are  oxamid,  malonamid,  and  biuret. 

To  establish  the  presence  of  proteid  all  of  these  tests  should  be  ap- 
plied since  any  one  of  them  may  be  given  by  substances  other 
than  proteids.  By  combining  color  reactions  with  some  of  the 
precipitation  reactions  given  below  conclusive  evidence  of  the  pres- 
ence of  proteid  can  be  obtained. 

Precipitation  Reactions. 

Coagulation  by  heating  and  precipitation  by  saturation  with  zinc 
or  ammonium  sulphate  have  been  mentioned  in  describing  the  dif- 
ferent groups.  It  should  be  noted  that  the  presence  of  other  nitro- 
gen compounds  including  urea,  pyridine,  and  piperidine  interferes 
with  the  coagulation  of  proteids.  \ 

Nitric  or  metaphosphoric  acid  precipitates  most  proteids  other 
than  peptones.  Potassium  ferrocyamde  precipitates  all  true  proteids 
other  than  peptones  from  solutions  acidulated  with  acetic  acid. 
Gelatin  is  not  precipitated.  Copper,  lead,  and  other  heavy  metals 
form  insoluble  compounds  with  proteids.  Basic  lead  acetate  is 
much  used  to  remove  proteids  from  solutions  to  be  examined  in  the 
polariscope  and  cupric  hydroxide  has  been  commonly  employed  for 
the  separation  of  proteids  from  amids  in  vegetable  foods.  Tannin  in 
the  presence  of  salt  precipitates  proteids,  including  peptones,  com- 
pletely (Allen).§  Phosphotungstic  acid  in  the  cold  precipitates  pro- 
teids and  simpler  nitrogen  compounds,  especially  the  diamino- 
acids ;  in  hot  solutions  small  amounts  of  peptones  and  simpler 
compounds  are  not  precipitated.  Bromine  added  to  saturation 
precipitates  proteids  including  peptones  and  gelatin.  According 
to  Allen  the  precipitation  is  quantitative. 

*  Neumeister  :  Ztschr.  Biol.,  1890,  26,  324. 

|  Ber.  deut.  chem.  Ges.,  1896,  29,  298;  Hammarsten-Mandel,  p.  31. 
J  Spiro  :  Ztschr.  physiol.  Chem.,  1900,  30,  182. 
$  The  completeness  of  the  precipitation  of  peptones  is  sometimes  questioned. 


206  ORGANIC   ANALYSIS. 

As  most  of  these  precipitation  reactions  are  also  given  by  alka- 
loids, and  some  of  them  by  amino-compounds,  it  is  important  in 
order  to  show  the  presence  of  proteids  to  precipitate  with  as  many 
reagents  as  possible.  The  precipitates  obtained  by  heat  coagula- 
tion and  by  acetic  acid  and  potassium  ferrocyanide  *  should  be 
tested  for  the  color  reactions  described  above.  For  other  precip- 
itation reactions  see  Halliburton :  Schafer's  Textbook  of  Physiology, 
I.,  Chapter  I.,  and  Cohnheim  :  Chemie  der  Eiweisskorper.  The 
completeness  of  precipitation  by  several  of  the  reagents  mentioned, 
and  the  interference  of  other  substances  are  discussed  in  the  papers 
cited  at  the  end  of  the  next  section. 

SEPARATION  OF  PROTEIDS  FROM  SIMPLER  NITROGEN  COMPOUNDS 
AND  FROM  EACH  OTHER. 

The  separation  of  proteids  from  simpler  nitrogen  compounds  is 
usually  based  upon  the  precipitation  of  the  former  by  some  reagent 
which  will  not  precipitate  the  other  nitrogen  compounds  present 
in  the  substance  under  examination.  The  choice  of  precipitant 
will  therefore  depend  upon  the  nature  of  the  substance  in  which 
proteid  is  to  be  determined.  Cupric  hydroxide  has  been  most 
commonly  used  for  vegetable,  and  tannin,  phosphotungstic  acid,  or 
bromine  for  animal  substances. 

The  methods  of  separating  proteids  from  each  other  are  based 
almost  entirely  upon  differences  in  solubilities  and  coagulation  or 
other  precipitation  reactions  such  as  have  been  given  in  character- 
izing the  groups  already  mentioned.  As  the  reactions  of  the  pep- 
tones approach  those  of  some  of  the  compounds  of  known  structure, 
the  separation  of  proteids  from  simpler  compounds  and  from  each 
other  can  best  be  studied  as  parts  of  the  same  problem.  The 
methods  available  for  these  separations  are  so  dependent  upon  the 
particular  combination  of  compounds  to  be  separated  that  any 
attempt  to  give  detailed  directions  would  be  of  less  value  than  refer- 
ences to  the  original  publications  among  the  more  important  of 
which  are  the  following :  — 

Stutzer:  Untersuchungen  ueber  die  quantitative  Bestimmung 
des  Proteinstickstoffes  und  die  Trennung  der  ProteinstofTe  von 
anderen  in  Pflanzen  vorkommenden  Stickstonrverbindungen,y<9//;';/. 
f.Landwirthschaft,  1880,28,  103;  1881,  29,  473  ;  1886,  34,  151. 

Schulze  und  Barbieri :    Zur  Bestimmung  des   Eiweisstoffe  und 

*  Winternitz  :  Ztschr.  physiol.  Chem.,  1892,  16,  439. 


PROTEIDS   AND    CEREALS.  207 

der  nichteiweissartigen  Stickstoffverbindungen  in  der  Pflanzen, 
Landw.  Vers.  Stat.,  26,  218. 

Teller:  The  Quantitative  Separation  of  Wheat  Proteids,  Bui.  42, 
Ark.  Agl.  Expt  Sta.,  p.  81  (1896). 

Allen  and  Searle:  Improved  Method  of  Determining  Proteid 
and  Gelatinoid  Substances,  Analyst,  1897,  22,  258. 

Baumann  und  Bomer :  Ueber  die  Fallung  der  Albumosen  durch 
Zinksulfat,  Ztschr.  Unters.  Nahr.-Genussm.,  1898,  i,  106. 

Mallet :  Analytical  Methods  for  Distinguishing  between  the 
Nitrogen  of  Proteids  and  that  of  the  Simpler  Amids  or  Amino- 
acids,  Bui.  54,  Div.  Chem.,  U.  S.  Dept.  Agriculture;  Chem.  News, 
1899,  80,  117,  168,  179. 

Vivian  :  A  Comparison  of  Reagents  for  Milk  Proteids,  i6th  Ann. 
Rpt.  Wis.  Agric'l.  Expt.  Sta.,  p.  179  (1899). 

Barnstein :  Ueber  eine  Modifikation  des  von  Ritthausen  vor- 
geschlagenen  Verfahrens  der  Eiweissbestimmung,  Landw.  Vers. 
Stat.,  1900,  54,  327  ;  Ztschr.  Unters.  Nahr.-Genussm.,  1901,  4,  688. 

Schjerning :  [A  series  of  papers  on  the  quantitative  separation 
and  precipitation  of  proteids],  Ztschr.  anal.  Chem.,  1894,  33,  263; 
I895.34,  135;  1896,35,  285;  1897,36,  643;  1898,37,  73,413; 
1900,39,  545,633. 

Fraps  and  Bizzell :  Methods  of  Determining  Proteid  Nitrogen 
in  Vegetable  Matter,  Journ.  Amer.  Chem.  Soc.,  1900,  22,  709. 

Hart :  Ueber  die  Quantitative  Bestimmung  der  Spaltungsprodukte 
von  Eiweisskorpern,  Ztschr.  physiol.  Chem.,  1901,  33,  347. 

Van  Slyke  and  Hart :  Methods  for  the  Estimation  of  the  Pro- 
teolytic  Compounds  contained  in  Cheese  and  Milk,  Bui.  215,  New 
York  Agl.  Expt.  Sta.,  Amer.  Chem.  Journ.,  1903,  29,  150. 

Bigelow :  Meat  and  Meat  Products,  U.  S.  Dept.  Agriculture, 
Bur.  Chem.,  Bui.  65,  pp.  10,  17;  Bui.  13,  Part  10,  p.  1396;  Bui. 
8 1,  p.  104. 

Grindley  :  A  Study  of  the  Nitrogenous  Constituents  of  Meats, 
U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  no,  Journ.  Amer. 
Chem.  Soc.,  1904,  26,  1086. 

Chamberlain  :  Determination  of  Gliadin  and  Gutenin  in  Flour, 
U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  118;  Circular  20, 
p.  14 

Grindley  and  Emmett :  The  Chemistry  of  Flesh,  Journ.  Amer. 
Chem.  Soc.,  1905,  27,  658. 


208  ORGANIC  ANALYSIS. 


CEREALS  AND  OTHER  GRAINS  — MILL  PRODUCTS. 

In  the  analysis  of  these  products  as  of  other  foods  and  feeding 
stuffs  of  vegetable  origin  the  constituents  usually  determined  are 
moisture,  fat,  proteid,  fiber,  and  ash.  The  remaining  constituents, 
estimated  by  difference,  constitute  the  so-called  nitrogen-free 
extract  composed  mainly  of  carbohydrates.  When  little  fiber  is 
present  its  determination  is  frequently  omitted,  in  which  case  fiber 
and  nitrogen-free  extract  are  reported  together,  usually  as  "  car- 
bohydrates by  difference."  When  a  further  examination  of  any 
one  of  these  groups  of  compounds  is  desired  it  can  be  made  ac- 
cording to  the  methods  already  described.  The  fat  obtained  by 
extraction  with  ether  can  be  studied  as  outlined  in  Chapters  IX.  to 
XL,  the  separation  of  the  nitrogen  compounds  has  been  discussed 
in  the  preceding  part  of  this  chapter,  and  methods  for  the  deter- 
mination of  the  principal  carbohydrates  are  given  in  Chapters  V.  to 
VII.  The  present  outline  will  therefore  be  confined  to  the  deter- 
minations first  mentioned.  Samples  for  analysis  should  be  air-dried 
and  ground  to  pass  a  sieve  of  one  half  millimeter  mesh,  though  for 
very  resistant  substances  a  sieve  having  round  holes  of  one  milli- 
meter diameter  may  be  used. 

DETERMINATION  OF  MOISTURE  AND  FAT. 

Dry  2  grams  for  5  hours,  or  to  constant  weight,  at  the  tempera- 
ture of  boiling  water,  if  possible  in  a  current  of  dry  hydrogen  or  in 
vacuo.  Consider  the  loss  of  weight  as  moisture.  Extract  the 
dried  sample  in  a  Soxhlet  or  continuous  extractor,  with  anhydrous 
alcohol-free  ether  for  sixteen  hours.  Dry  the  extract  to  constant 
weight  in  a  boiling  water  oven.  The  ether  extraction  should  be 
carried  out  at  a  distance  from  any  free  flame,  the  flask  being  heated 
by  a  safety  water  bath  or,  more  conveniently,  by  an  electric  heater. 

Notes.  —  The  ether  extract  of  vegetable  substances  often  con- 
tains in  addition  to  fat  more  or  less  of  coloring  matters  and  resin- 
ous substances,  these  being  more  readily  soluble  in  ether  containing 
fatty  oils  than  in  ether  alone.  In  the  cereal  grains  and  especially 
in  the  milling  products  from  which  the  outer  layers  of  the  grains 
have  been  separated  the  amount  of  such  impurities  is  usually  negli- 
gible. If  the  ether  extract  is  made  to  percolate  a  layer  of  animal 
charcoal,  practically  pure  fat  is  obtained.* 

*  Patterson  :  Amer.  Chem.  Journ.,  12,  261. 


PROTEIDS   AND    CEREALS.  209 

The  reason  for  drying  in  a  current  of  hydrogen  rather  than  in 
air  is  that  the  oils  of  the  cereal  grains  belong  to  the  "  semidrying" 
group  and  therefore  absorb  oxygen  when  exposed  to  air,  especi- 
ally at  high  temperature.  This  will  of  course  increase  the  weight 
of  the  fat  and  make  the  apparent  percentage  of  moisture  too  low. 
The  partially  oxidized  oils  are  also  apt  to  be  incompletely  ex- 
tracted by  ether.  For  a  full  discussion  of  the  determination  of 
water  in  foods  and  physiological  preparations,  see  Benedict  and 
Manning:  Amer.  Jonrn.  PhysioL,  1905,  13,  309. 

Fat  may  also  fail  of  complete  extraction,  even  when  unchanged, 
by  being  occluded  or  mechanically  enclosed  in  carbohydrate  or 
proteid  material  which  is  impervious  to  the  ether. 

Although  ether  extracts  may  be  evaporated  at  the  temperature 
of  boiling  water  without  loss  of  fat,  such  loss  has  been  found  to 
occur  in  drying  moist  samples  even  in  a  current  of  hydrogen.  That 
the  loss  in  such  cases  is,  in  part  at  least,  due  to  an  actual  volatili- 
zation of  material  may  be  shown  by  passing  the  current  of  hydro- 
gen in  which  the  sample  is  dried  into  strong  sulphuric  acid.  This 
loss  is  probably  due  to  the  action  of  the  escaping  steam  and  may 
be  practically  avoided  by  drying  at  a  lower  temperature,  preferably 
in  a  partial  vacuum. 

All  three  of  the  causes  just  mentioned  tend  toward  a  deficiency 
of  fat  in  the  analysis  of  cooked  foods  prepared  from  cereal  products. 
Thus  in  a  number  of  experiments  on  bread  making  *  the  fat  found 
by  analysis  of  the  dried  bread  was  less  than  half  of  that  contained 
in  the  materials  used  and  the  iodine  figure  of  the  ether  extract  of 
the  bread  was  only  60.4  as  against  101.4  in  that  of  the  original 
flour  showing  that  a  very  considerable  oxidation  had  taken  place 
even  in  that  portion  of  the  fat  which  was  still  soluble  in  ether. 

Berntrop's  method  for  the  determination  of  fat  in  breadstuff's  is  as 
follows  :  f  Mix  150  grams  of  fresh  bread  with  500  c.c.  of  water,  add 
100  c.c.  of  concentrated  hydrochloric  acid  and  boil  for  two  hours 
connected  with  a  reflex  condenser.  J  Cool  the  resulting  brown 
liquid  to  room  temperature,  filter  with  suction  through  a  mois- 
tened fat-free  paper,  and  wash  with  cold  water.  Dry  the  paper  and 
residue  for  an  hour  at  100°  to  110°,  remove  the  residue  as  com- 

*  Bui.  67,  Office  of  Experiment  Stations,  U.  S.  Dept.  Agriculture, 
f  Ztschr.  angrw.  Ghent.,  1902,  1 21. 

J  In  treating  meal  or  flour,  heat  for  an  hour  in  a  waterbath  and  then  boil  for  an  hour 
with  the  reflux  condenser  attached. 


210  ORGANIC   ANALYSIS. 

pletely  as  possible  from  the  filter  paper  and  grind  it  with  sand  in  a 
mortar.  Cut  up  and  add  the  filter  paper,  and  transfer  the  dry 
mixture  to  a  paper  extraction  thimble  and  treat  with  ether  or  petro- 
leum ether  in  an  extractor. 

Dormeyet's  method*  designed  originally  for  the  determination  of 
fat  in  animal  tissues  has  been  adapted  to  vegetable  foods  by 
Beger.*  From  3  to  5  grams  of  substance  are  mixed  with  480  c.c. 
of  water,  20  c.c.  of  25  per  cent,  hydrochloric  acid,  and  one  gram 
of  fat-free  pepsin.  The  mixture  is  kept  at  37°  to  40°  for  twenty- 
four  hours,  filtered  with  suction  through  a  paper  supported  on  a 
porcelain  plate  and  covered  with  asbestos,  and  both  the  filtrate  and 
the  residue  extracted  with  ether. 


DETERMINATION  OF  NITROGEN  COMPOUNDS. 

Determine  total  nitrogen  as  described  in  Chapter  II.  For  the 
separation  of  proteid  from  non-protied  nitrogen  in  cereals. 
Stutzer's  cupric  hydroxide  method;);  has  been  most  commonly 
used  and  is  "  official "  for  agricultural  products.  In  examining 
wheat  flour  with  reference  to  its  baking  qualities  it  is  desirable  to 
determine  also  the  salt-soluble  and  the  alcohol -soluble  proteids 
using  the  methods  recently  adopted  provisionally  by  the  Official 
Agricultural  Chemists. § 

The  extended  investigations  by  Osborne  and  his  associates  ||  and 
by  Ritthausen  \  have  shown  that  nearly  all  of  the  cereal  proteids 
contain  over  16  per  cent,  of  nitrogen  so  that  the  results  obtained 
by  multiplying  the  nitrogen  by  6.25  are  too  high.  The  factors  now 
regarded  as  most  nearly  correct  are ;  for  wheat,  rye,  and  barley, 
5.70  **  ;  for  maize,  oats,  rice,  and  buckwheat,  6.00.  The  old  factor 
6.25  is,  however,  still  frequently  used  for  the  sake  of  uniformity  or 
for  comparison  with  earlier  work.  In  reporting  results,  therefore, 
the  factor  used  should  always  be  given. 

*Arch.f.ges.  PhysioL  (Pfluger],  1895,  61,  341  5   1896,  65,  90. 

f  Chem.  Ztg.,  1902,  26,  112. 

t  Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

§  Circular  20,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

||  Reports  Conn.  Agl.  Expt.  Station,  1890,  et  seq.  Much  of  the  work  has  also 
appeared  in  Journ.  Amcr.  Chem.  Soc.,  Amer.  Chem.  Journ.  ^  and  Amer.  Journ. 
PhysioL 

\  Summarized  in  Landw.   Vers.  Stat.,  1896,  47,  391. 
**  The  factor  5.68  has  recently  been  proposed  for  wheat  flour. 


PROTEIDS   AND    CEREALS.  211 

DETERMINATION  OF  CRUDE  FIBER.* 

Extract  2  grams  of  the  substance  with  ordinary  ether  or  use  the 
residue  from  the  determination  of  the  ether  extract.  To  this 
residue,  in  a  500  c.c.  flask,  add  200  c.c.  of  boiling  1.25  per  cent, 
sulphuric  acid ;  connect  the  flask  with  an  inverted  condenser,  the 
tube  of  which  passes  only  a  short  distance  beyond  the  rubber 
stopper  into  the  flask.  Boil  at  once,  and  continue  the  boiling  for 
thirty  minutes.  A  blast  of  air  conducted  into  the  flask  may  serve 
to  reduce  the  frothing  of  the  liquid.  Filter,  wash  with  boiling 
water  till  the  washings  are  no  longer  acid ;  rinse  the  substance 
back  into  the  same  flask  with  200  c.c.  of  a  boiling  1.25  per  cent, 
solution  of  sodium  hydroxide,  practically  free  from  sodium  car- 
bonate; boil  at  once,  and  continue  the  boiling  for  thirty  minutes 
in  the  same  manner  as  directed  above  for  the  treatment  with  acid. 
Filter  on  a  Gooch  crucible,  and  wash  with  boiling  water  till  the 
washings  are  neutral;  dryatno0;  weigh;  incinerate  completely. 
The  loss  of  weight  is  crude  fiber. 

The  filter  used  for  the  first  filtration  may  be  linen,  one  of  the  forms 
of  glass  wool  or  asbestos  filters,  or  any  other  form  that  secures  clear 
and  reasonably  rapid  filtration.  The  solutions  of  sulphuric  acid 
and  sodium  hydroxide  are  to  be  made  up  of  the  specified  strength, 
determined  accurately  by  titration  and  not  merely  from  specific 
gravity. 

DETERMINATION  OF  ASH. 

Char  about  2  grams  and  burn  to  whiteness  at  the  lowest  pos- 
sible red  heat,  preferably  in  a  flat  bottomed  platinum  dish  in  a 
muffle. 

If  considerable  quantities  of  phosphates  are  present  these  may 
fuse  over  some  of  the  carbon  and  render  its  combustion  very  slow. 
In  such  cases,  extract  the  charred  mass  with  a  little  hot  acetic  acid, 
set  aside  the  solution  till  the  char  is  burned,  then  evaporate  it  to 
dryness  in  the  same  dish  and  heat  the  residue  to  dull  redness  till 
the  ash  is  white  or  nearly  so.  Samples  containing  added  salt  should 
be  extracted  with  water  before  charring  and  the  determination 
finished  as  just  described.  The  precautions  given  in  Chapter  I. 
should  always  be  observed. 

*  Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture.  See  also  Thatcher  :  Journ.  Amer. 
Chem.  Soc.,  1902,  24,  I2IO;  Browne  :  Ibid^  1904,  25,  315. 


212  ORGANIC   ANALYSIS. 

ADDITIONAL  TESTS  AND   DETERMINATIONS. 

For  discussions  of  tests  for  foreign  substances  present  as  adulter- 
ants the  references  given  below  should  be  consulted.  Such  adul- 
teration of  cereal  products  is  not  common  in  America.  The  deter- 
minations most  often  required,  in  addition  to  those  already  given 
are  such  as  to  show  the  usefulness  of  the  material  for  some  partic- 
ular purpose.  The  value  of  wheat  flour  for  breadmaking  depends 
largely  upon  the  proportion  of  alcohol-soluble  proteid  (gliadin) 
which  it  contains,  and  upon  the  acidity.  The  gliadin  is  most 
abundant  in  the  interior  of  the  wheat  kernel  and  therefore  in  the 
highest  commercial  grades  of  flour.  Acidity  is  objectionable  both 
as  an  indication  of  deterioration  and  because  it  acts  upon  the  gli- 
adin injuring  the  physical  properties  which  are  especially  impor- 
tant in  breadmaking. 

To  determine  acidity,  shake  10  grams  of  the  dry  sample  with 
IOO  c.c.  of  cold  water,  filter,  and  titrate  an  aliquot  part  with  tenth- 
normal  sodium  or  potassium  hydroxide  using  phenolphthalein  as 
indicator.  In  fine  flour  the  acidity  calculated  as  lactic  acid  should 
not  exceed  o.io  per  cent. 

INTERPRETATION  OF  RESULTS.   ' 

Official  Definitions  and  Standards* 

1.  Grain  is  the   fully   matured,    clean,    sound,  air-dry  seed  of 
wheat,  maize,  rice,  oats,  rye,  buckwheat,  barley,  sorghum,  millet,  or 
spelt. 

2.  Meal  is  the  sound  product  made  by  grinding  grain. 

3.  Flour  is  the  fine,  sound  product  made  by  bolting  wheat  meal 
and  contains  not   more  than  13.5  per  cent,  of  moisture,  not  less 
than  1.25  per  cent,  of  nitrogen,  not  more  than  i.o  per  cent,  of  ash, 
and  not  more  than  0.50  per  cent,  of  fiber. 

4.  Graham  flour  \s  unbolted  wheat  meal. 

5.  "  Whole    wheat  flour"    "entire  wheat  flour"    improperly   so 
called,  is  fine  wheat  meal  from  which  a  part  of  the  bran  has  been 
removed. 

6.  Gluten  flour  is  the  product  made  from  flour  by  the  removal  of 
starch  and  contains  not  less  than  5.6  per  cent,  of  nitrogen  and  not 
more  than  10  per  cent,  of  moisture. 

7.  Maize  meal,  corn  meal  or  Indian  corn  meal  is  meal  made  from 
sound  maize  grain  and   contains  not   more  than   14  per  cent,  of 
moisture,  not  less  than   1.12  per  cent,  of  nitrogen,  and  not  more 
than  1.6  per  cent  of  ash. 

8.  Rice-\s  the  hulled  and  polished  grain  of  Oryza  sativa. 

9.  Oatmeal  is  meal  made  from  hulled  oats,  and  contains  not  more 

*  Circular  No.  13,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 


PROTEIDS   AXD    CEREALS. 


213 


than  8  per  cent,  of  moisture,  nor  more  than  1.5  per  cent,  of  crude 
fiber,  not  less  than  2.24  per  cent,  of  nitrogen,  and  not  more  than 
2.2  per  cent,  of  ash. 

10.  Rye  flour  is  the  fine  sound  product  made  by  bolting  rye  meal 
and  contains  not   more  than    13.5   per  cent,  of  moisture,  not  less 
than  1.36  per  cent,  of  nitrogen,  and  not  more  than    1.25  per  cent, 
of  ash. 

11.  Buckivh  eat  flour  is  bolted  buckwheat  meal  and  contains  not 
more  than  12  per  cent,  of  moisture,  not  less  than  1.28  per  cent,  of 
nitrogen,  and  not  more  than  1.75  per  cent,  of  ash. 

Where  percentages  are  given   in   these  standards  they  are,  of 
course,  intended  to  represent  normal  limits  rather  than  averages  or 

extreme  limits. 

Composition  of  Entire  Grains. 

Wiley*    estimates    the    approximate   composition   of    average 
typical  American  grains  as  follows: 

PERCENTAGE  COMPOSITION  OF  ENTIRE  GRAINS  (WILEY). 


Barley. 

Buck- 
wheat. 

Maize. 

Oats. 

Rice 

unhulled. 

Rye. 

Wheat. 

Moisture         

10.85 

12.  OO 

10.71; 

IO.OO 

IO.5O 

10.50 

1  0.60 

Protein  (Nitrogen  X6-25)  
Fat  (Ether  Extract) 

II.  OO 

2.  25 

iQ-75 

2  OO 

IO.OO 
4  25 

12.  OO 
4-  5O 

7<5Q 
i.  60 

12.25 
I.5O 

12.25 
1-75 

Crude  Fiber 

3.85 

IO.  7? 

I.7C 

I2.OO 

Q.OO 

2.  IO 

2  40 

Ash  

2.50 

1.75 

1.50 

3.50 

4.OO 

I.QO 

1.75 

Carboh  vd  rates  (d  iff.)  .. 

60.45 

62  75 

71.75 

58.00 

67.40 

71-75 

71-25 

Composition  of  Mill  Products. 

An  extended  study  of  the  mill  products  of  wheat  made  by  Teller 
at  the  Arkansas  Experiment  Station,  1894  to  1898,-)-  included  a 
milling  experiment  in  which  the  principal  products  of  a  long  proc- 
ess (/  break)  roller  mill  were  analyzed  with  the  following  results  : 

PERCENTAGE  COMPOSITION  OF  MILL  PRODUCTS  OF   WHEAT  (TELLER). 


Patent 
Flour. 

Straight 
Flour. 

Low 
Grade 
Flour. 

Ship 
Stuff. 

Bran. 

Whole 
Wheat. 

Pure 
Germ. 

Moisture 

13-75 

I  7.QO 

I  3.22 

12  25 

12.85 

I3.QO 

6.80 

Ash 

.  33 

•  47 

,QO 

3  12 

« 
5.80 

2.15 

4.65 

Crude  Fiber                

.17 

.26 

•  74 

3.55 

6.14 

2.17 

1.  60 

Fat                         

1.05 

1.25 

I.7O 

4.80 

5.20 

2.15 

14.38 

Protein  (Nitrogen  X  5-7)  

*   •> 
0.60 

10.37 

12.88 

16.36 

15.56 

12.31 

36.00 

Carbohydrates  (  diff.  )  

75.01 

73-75 

70.56 

5Q.O2 

54-45 

63.32 

/• 

36.55 

Total  Nitrogen  

1.70 

1.82 

2.26 

2.87 

2.73 

2.16 

6-34 

1.65 

1.72 

2.  2O 

2  68 

2  51 

1.98 

Amid  Nitroffen... 

.0; 

.10 

.06 

.10 

.22 

.18 

t 

*Bull.  45,  Bur.  Chem.  U.  S.  Dept.  Agriculture. 

f  Buls.  42  and  53,  Ark.  Expt.  Station  (Fayetteville,  Ark.). 

J  The  germ  is  richer  in  amid  nitrogen  than  other  parts  of  the  wheat  kernel. 


214  ORGANIC   ANALYSIS. 

Under  the  system  of  milling  now  practiced  in  the  Northwest  a 
number  of  "  streams  "  of  flour  are  produced  which  are  afterward 
united  in  different  proportions  to  form  the  market  grades  of  flour. 
Snyder  has  recently  published*  analyses  of  the  different  "  streams  " 
as  obtained  in  milling  No.  I  Northern  wheat  by  typical  modern 
machinery.  A  sample  of  wheat  containing  13.07  per  cent,  mois- 
ture and  2.28  per  cent,  nitrogen  gave  streams  of  flour  containing 
from  9.01  to  11.68  per  cent,  moisture,  and  from  1.92  to  2.95  per 
cent,  of  nitrogen.  The  "  gliadin  number,"  or  percentage  of  the 
total  nitrogen  existing  in  the  form  of  alcohol-soluble  proteids, 
varied  from  39.51  to  66.67.  It  ls  interesting  to  note  that  some  of 
the  streams  of  flour  thus  obtained  from  average  wheat  in  the  ordi- 
nary milling  process  contain  considerably  more  nitrogen  than  was 
recently  found  in  the  majority  of  so-called  gluten  and  diabetic  flours 
obtained  in  the  market,  f 

For  additional  analyses  and  results  of  experiments  upon  the 
digestibility  and  nutritive  value  of  cereal  products  see  Bui.  13,  Part 
9,  Bureau  of  Chemistry,  and  Buls.  28,67,  85,  101,  126,  and  143, 
Office  of  Experiment  Stations,  U.  S.  Department  of  Agriculture. 

REFERENCE  BOOKS. 

Allen :  Commercial  Organic  Analysis,  Volume  IV. 

Cohnheim:  Chemie  der  Eiwisskorper  (2d  Ed.),  Braunschweig, 
1904. 

Hammarsten :  Physiological  Chemistry  (Trans,  by  Mandel),  New 
York,  1904. 

Hoppe-Seyler  and  Thierfelder :  Physiologisch-  und  Pathologisch 
Chemischen  Analyse. 

Konig :  Die  Menschliche  Nahrungs-  und  Genussmittel  (4th  Ed.). 

Leach:  Food  Inspection  and  Analysis,  New  York,  1904. 

Maurizio :  Getreide,  Mehl  und  Brot,  Berlin,  1903. 

Neumeister :  Lehrbuch  der  Physiologische  Chemie,  1897. 

Schafer:  Textbook  of  Physiology,  Volume  I.,  London,  1898. 

Vogl :  Die  wichtigsten  vegetabilischen  Nahrungs-  und  Genuss- 
mittel, Leipzig,  1899. 

Wiley :  Agricultural  Analysis,  Volume  III.;  U.  S.  Dept.  Agri- 
culture, Bur.  Chem.,  Bui.  13,  Part  IX. 

Vereinbarungen  zur  einheitlichen  Untersuchung  und  Beurthei- 
lung  von  Nahrungs-  und  Genussmitteln  fur  das  Deutsche  Reich. 
Heft  II.  Berlin,  1899. 

*  Bui.  85,  Minn.  Expt.  Station,  St.  Anthony  Park,  Minn.,  1904. 

f  Sherman  and  Burr  :   New  York  Medital  Journal,  74,  686  (Oct.  12,  1901). 


CHAPTER   XIV. 

Milk. 

Cows'  milk  is  concisely  described  as  essentially  an  aqueous  solu- 
tion of  milk  sugar,  albumin,  and  certain  salts,  holding  in  suspen- 
sion globules  of  fat  and  in  a  state  of  semi-solution  casein  together 
with  mineral  matter  (Richmond).  Small  amounts  of  other  com- 
pounds are  also  present,  but  need  not  be  considered  here. 

Standard  milk  (whole  milk)  is  defined  *  as  the  lacteal  secretion 
obtained  by  the  complete  milking  of  one  or  more  healthy  cows 
properly  kept  and  fed,  excluding  that  obtained  within  fifteen  days 
before  and  five  days  after  calving,  and  contains  not  less  than  12 
per  cent,  of  total  solids,  not  less  than  8.5  per  cent,  of  solids  not  fat, 
and  not  less  than  3.25  per  cent,  of  milk  fat. 

These  limits  are  considerably  below  the  average  and  consider- 
ably above  the  lowest  authentic  figures  which  have  been  found. 
Average  milk  may  be  assumed  to  contain  12.9  to  13  per  cent,  of 
total  solids  made  up  of :  — 

Fat.  Protein.  Milk  Sugar. f  Ash. 

In  round  numbers  I   4.0  3.3  5.0  0.7 

Estimated  average   4.00  3.35  4.88  0.72 

The  protein  content  of  average  milk  is,  therefore,  about  one- 
fourth  of  the  total  solids.  In  general  the  same  relation  holds  in 
milk  which  is  richer  than  the  average.  Each  increase  of  I  per  cent, 
in  total  solids  thus  involves  on  the  average  an  increase  of  0.25  per 
cent,  of  proteids,  the  remaining  0.75  per  cent,  being  practically  all 
fat.  This  increase  in  proteids  and  fat  is  usually  accompanied  by  a 
slight  increase  in  ash  and  decrease  in  milk  sugar.  The  following 
average  percentages  illustrate  these  relations  in  rich  milk :  — 

Total  Solids.  Fat.  Protein.  Milk  Sugar.  Ash. 
Average  for  5  years  ;  mixed  evening  milk 

of  400  to  500  cows 14.62  5.39  3.66  4.82  0.75 

Average  of  13  unusually  rich  samples  from 

individual  cows 18.03         7-?6  4-68         4.76        0.83 

*  Circular  No.  13,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture, 
f  The  figures  for  milk  sugar  include  the  small  amount  of  undetermined  non-nitrog- 
enous matter. 

+  These  are  the  figures  used  in  most  publications  of  the  U.  S.  Dept.  Agriculture. 

215 


216 


ORGANIC   ANALYSIS. 


The  composition  of  milk  of  less  than  average  richness  cannot  be 
so  definitely  stated.  In  some  cases  there  is  a  deficiency  of  fat  and 
protein  with  no  decrease  in  milk  sugar,  while  in  other  cases  the 
reverse  is  true.  Usually  if  an  unadulterated  milk  is  poor  in  fat  it 
will  be  found  proportionately  poor  in  protein,  while  if  the  fat  is 
normal  the  protein  is  usually  also  normal  and  the  low  percentage 
of  solids  not  fat  is  due  to  a  deficiency  in  milk  sugar. 

Several  hundred  American  analyses,  made  before  1890,  compiled 
and  averaged  in  ten  groups  arranged  according  to  percentage  of 
total  solids,  gave  the  following  results.* 

COOKE'S  COMPILATION  OF  AMERICAN  ANALYSES  OF  MILK. 


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As  a  rule  the  percentage  of  milk  sugar  and  ash  is  most  nearly 
constant,  that  of  fat  is  most  variable,  while  the  protein  varies  with 
the  fat,  but  to  a  much  smaller  extent.  The  variations  which  may 
be  regarded  as  usual  and  the  extreme  variations  which  the  writer 
has  found  authentically  recorded  are  as  follows : — 


Fat. 
Per  Cent. 

Usual  variations 3-6 

Extreme  variations f  ..  1.04-14.67 


Solids  Not  Fat. 
Per  Cent. 


Proteids. 
Per  Cent 


Milk  Sugar. 
Per  Cent. 


Ash. 
Per  Cent 


0.7-0.78 
0.66-1.44 


8.5-9-5  3-4  4-6-5 

4.90-13.76        2.86-9.98        2.33-5.28 

The  extreme  variations  are  of  no  practical  value  as  a  means  of 
determining  the  limits  within  which  milk  shall  be  considered  un- 
adulterated, partly  because  it  is  possible  to  practice  "  adulteration 
through  the  cow  "  (z.  e.t  by  selection,  feeding,  and  manner  of  milk- 
ing to  obtain  "  genuine "  milk  much  below  the  normal  quality), 
but  mainly  because  the  milk  which  reaches  market  is  practically 
always  the  mixed  product  of  several  cows  so  that  individual  varia- 
tions have  comparatively  little  effect. 

There  are  many  causes  of  variation  in  the  composition  of  cow's 
milk.  Only  the  most  important  can  be  given  here.  Other  con- 

*Cooke  :  Vermont  Agricultural  Experiment  Station  Report  for  1890,  p.  97. 
f  Including  only  results  obtained  from  apparently  healthy  cows,  believed  to  have 
been  milked  regularly  under  normal  conditions. 


MILK.  217 

ditions  being  normal  the  percentages  of  fat  and  proteids  are  higher 
in  autumn  and  winter  than  in  spring  and  summer;  they  also  in- 
crease as  the  amount  of  milk  decreases  toward  the  end  of  each 
period  of  lactation.  Milk  drawn  in  the  evening  is  generally  0.3  to 
0.4  per  cent,  richer  in  fat  than  that  obtained  in  the  morning,  and 
at  any  one  milking  the  last  portions  drawn  are  much  richer  than 
the  first.  The  influence  of  a  change  of  food  upon  the  percentage 
composition  of  milk  is  usually  only  temporary.  In  general  the 
peculiarities  of  breed  *  and  the  qualities  of  individual  animals  are 
the  most  important  factors  in  determining  the  richness  of  milk. 
Aside  from  all  these  conditions  the  milk  of  individual  cows  is  sub- 
ject to  considerable  fluctuation,  especially  in  fat  content.  Thus 
the  analyses  of  60  monthly  samples  of  the  mixed  milk  of  about 
500  cows  showed  a  variation  of  0.89  in  the  percentage  of  fat,  the 
greatest  deviation  from  the  average  being  0.50  per  cent.  About 
one-half  of  the  determinations  of  fat  in  the  milk  of  individual  cows 
of  the  herd  during  the  same  period  were  more  than  0.50  per  cent, 
and  about  one-fifth  were  more  than  i.o  per  cent,  above  or  below 
the  average.  Milk  representing  the  mixed  product  of  several 
farms,  such  as  is  now  commonly  sold  in  large  cities,  should,  there- 
fore, be  much  more  uniform  in  composition  than  that  of  a  single 
cow  or  a  small  herd.f 

TAKING  AND  PRESERVATION  OF  SAMPLES. 
.If  the  lot  of  milk  to  be  sampled  is  small  it  can  be  mixed  by 
pouring  from  one  vessel  to  another  from  two  to  ten  times,  accord- 
ing to  the  extent  to  which  the  cream  has  separated,  and  the  por- 
tion for  analysis  dipped,  or  withdrawn  by  means  of  a  pipette,  from 
near  the  center.  When  the  sample  is  too  large  to  be  handled  in 
this  way  it  should  be  transferred  if  necessary  to  a  cylindrical  can 
and  sampled  by  means  of  a  Scovell  tube.J  In  order  to  obtain  a 

*For  comparison  of  the  milk  of  different  breeds  see  Richmond's  Dairy  Chemistry, 
pp.  122-126,  Report  of  the  New  York  State  Expt.  Station  for  1891  (abstracted  in  the 
Expt.  Station  Record,  4,  263),  and  Report  of  the  Wisconsin  Expt.  Station  for  1901, 

P-  85- 

|  Fuller  discussions  of  the  variations  in  the  milk  of  individual  cows  and  mixed 
milk  of  herds  will  be  found  in  some  of  the  reference  books  given  at  the  end  of  the 
chapter,  in  the  papers  on  the  composition  of  milk  published  annually  by  Richmond  in 
the  Analyst,  and  in  Hittcher's  Gesammtbericht  tiber  die  Untersuchung  der  Milch, 
Berlin,  1899. 

| Wiley's  Agricultural  Analysis,  Vol.  III.,  p.  470,  Leach's  Food  Inspection  and 
Analysis,  p.  95.  These  sampling  tubes  are  sold  by  dealers  in  dairy  apparatus. 


218  ORGANIC   ANALYSIS. 

proper  sample  of  a  large  lot  of  milk  delivered  in  cans  of  the  same 
diameter,  it  is  only  necessary  to  sample  each  can  with  the  Scovell 
tube  and  mix  the  portions  thus  obtained. 

The  sample  should  be  placed  at  once  in  a  clean,  dry,  sterile 
bottle,  tightly  stoppered,  and  analyzed  as  soon  as  possible.  Before 
withdrawing  each  portion  for  analysis  the  sample  must  be 
thoroughly  mixed  by  pouring  —  not  by  shaking. 

If  the  analysis  cannot  be  made  at  once  or  if  the  sample  is  to  be 
kept  for  some  time  after  the  analysis,  it  must  either  be  stored  at  a 
temperature  near  the  freezing  point  or  preserved  by  the  addition 
of  an  antiseptic.  Formaldehyde  *  added,  while  the  milk  is  still 
fresh,  in  the  proportion  of  I  :  1000  will  preserve  the  sample  for 
months  without  apparent  change.  This  amount  of  formaldehyde 
has  a  scarcely  perceptible  influence  upon  the  analytical  results. 
If  preservation  for  only  a  few  days  is  required  a  smaller  amount  of 
formaldehyde  should  be  used,  I  :  2000  to  i  :  10,000  according  to 
the  freshness  of  the  milk. 

PRELIMINARY  OR  PARTIAL  EXAMINATION. 
DETERMINATION  OF  SPECIFIC  GRAVITY. 

The  specific  gravity  of  milk  is  usually  between  1.029  and  1.034. 
Since  cream  is  considerably  lighter  than  milk,  the  specific  gravity 
would  be  lowered  by  the  addition  of  water  or  of  cream,  but  cases 
in  which  genuine  milk  shows  a  low  specific  gravity  as  a  result  of 
high  fat  content  are  very  rare.  As  already  explained,  high  per- 
centages of  fat  are  normally  accompanied  by  high  percentages  of 
proteids,  so  that  in  most  cases  the  specific  gravity  is  higher  in  rich 
than  in  poor  milk.  With  practice  the  samples  which  are  excep- 
tions to  this  rule  can  usually  be  detected  by  noticing  the  apparent 
viscosity  and  opacity  of  the  milk  as  it  runs  from  the  surface  of  the 
lower  bulb  of  the  lactometer.  The  specific  gravity  taken  in  con- 
nection with  this  appearance  is  much  used  as  a  preliminary  test  by 
milk  inspectors  and  is  recommended  by  Richmond  as  the  best 
means  of  rapidly  testing  each  lot  of  milk  received^by  a  large  dairy. 

The  Quevene,  Veith,  and  Soxhlet  lactometers  are  hydrometers 
of  sufficient  range  for  use  with  milk  and  so  graduated  as  to  read 
the  "  excess  gravity"  over  water  taken  as  1000.  Thus  a  milk  of 

*  Other  preservatives  are  sometimes  useful.  See  Richmond's  Dairy  Chemistry,  p. 
144. 


MILK.  219 

1.0315  specific  gravity  gives  a  lactometer  reading  of  31.5°.  These 
instruments  are  often  made  to  include  a  Fahrenheit  thermometer, 
the  scale  of  the  latter  being  on  the  same  stem  with  the  lactometer 
scale.  The  lactometer  reading  should  be  taken  between  50°  and 
65°  F.  and  corrected  for  temperature  by  adding  or  subtracting 
0.1°  for  each  degree  F.  above  or  below  60.  The  New  York  Board 
of  Health  lactometer  has  a  scale  reading  zero  in  pure  water  and 
roo  at  1.029  specific  gravity.  To  convert  the  readings  of  this  scale 
into  the  lactometer  degrees  described  above,  multiply  by  0.29. 

VOLUMETRIC  DETERMINATION  OF  FAT. 

Babcock,  in  1890,  introduced  the  first  satisfactory  rapid  method 
for  the  determination  of  fat  in  milk.  On  mixing  milk  with  ap- 
proximately an  equal  volume  of  strong  sulphuric  acid,  the  casein 
is  dissolved  while  the  fat  remains  unchanged  and  can  be  sepa- 
rated by  centrifugal  force.  The  test  is  performed  in  a  bottle  with 
a  neck  so  graduated  that  the  percentage  of  fat  can  be  read  off 
directly  upon  removing  the  bottle  from  the  centrifuge. 

Determination.  —  Measure  17.6  c.c.  of  milk  at  14°  to  18°  (about 
55°  to  65°  F.)  and  introduce  into  the  test  bottle.  Add  17.5  c.c.  of 
sulphuric  acid  of  1.82  sp.  gr.  (commercial  concentrated  acid  is 
usually  the  right  strength)  allowing  the  acid  to  flow  down  the  side 
of  the  bottle  so  as  not  to  mix  with  the  milk.  When  acid  has  been 
added  to  all  of  the  bottles  and  everything  is  ready  to  start  the 
whirling,  mix  the  milk  and  acid  quickly  and  thoroughly  by  shak- 
ing and  continue  the  shaking  until  the  curd  is  in  solution  and  the 
liquid  has  reached  a  permanent  and  uniform  color.  Then  place  an 
even  number  of  the  bottles  in  opposite  pockets  in  the  machine 
and  whirl  at  the  rate  of  800  to  1200  revolutions  per  minute, 
according  to  the  diameter  of  the  wheel  which  carries  the  bottle. 
After  whirling  five  minutes,  fill  with  hot  water  to  the  shoulder  of 
the  bottle  and  whirl  two  minutes,  then  fill  with  hot  water  to  near 
the  top  of  the  graduation  in  the  neck  and  whirl  again  for  two 
minutes.  The  percentage  of  fat  is  now  shown  by  the  height  of 
the  column  in  the  graduated  neck  of  the  bottle. 

Notes.  —  The  capacity  of  the  graduated  neck  of  the  bottle  from 
o  to  10  is  2  c.c.  It  is  assumed  that  the  17.6  c.c.  of  milk  taken  for 
the  determination  will  weigh  ten  times  as  much  as  2  c.c.  of  warm 
butter  fat.  It  is  important  that  the  final  readings  be  taken  while 
the  fat  is  still  warm.  On  account  of  the  unavoidable  contraction 


220  ORGANIC   ANALYSIS. 

of  the  fat  while  taking  these  readings  it  is  customary  to  read  from 
the  bottom  of  the  lower  to  the  top  of  the  upper  meniscus.  The 
result  is  usually  within  0.2  per  cent,  of  that  found  by  the  gravi- 
metric method.  The  column  of  fat  should  be  of  a  clear  yellow 
color  throughout.  If  the  acid  used  is  too  weak,  flocks  of  undis- 
solved  casein  are  apt  to  be  found  in  the  lower  part  of  the  fat  col- 
umn ;  if  too  strong  the  acid  may  char  the  fat.  For  a  full  discus- 
sion of  the  details  of  the  test  with  directions  for  applying  it  to  other 
dairy  products  see  the  work  of  Farrington  and  Woll.* 

The  most  important  modifications  of  the  Babcock  method  are 
fully  described  in  Richmond's  Dairy  Chemistry,  pp.  1/4-192. 

CALCULATION  OF  SOLIDS  FROM  SPECIFIC  GRAVITY  AND  FAT. 

Many  formulae  have  been  proposed  by  which  to  calculate  the 
percentage  of  solids  in  milk  from  the  percentage  of  fat  and  the 
specific  gravity.  The  results  thus  obtained  are  sufficiently  accurate 
for  many  technical  purposes  and  often  for  routine  inspection  work 
which  is  not  to  be  made  the  basis  of  legal  action.  Such  formulae 
may  be  found  in  many  of  the  works  referred  to  at  the  end  of  this 
chapter.  They  are  necessarily  based  on  the  assumption  that  each 
per  cent,  of  fat  causes  a  definite  decrease,  and  each  per  cent,  of 
solids  not  fat  a  definite  increase,  in  the  specific  gravity. 

Since  the  solution  densities  of  proteids,  milk  sugar,  and  milk  ash 
differ  considerably  f  any  change  in  the  relative  proportions  of  these 
constituents  must  alter  the  solution  density  of  the  solids  not  fat 
and  thus  diminish  the  accuracy  of  this  method. 

One  of  the  simplest  of  these  formulae  is  that  of  Richmond : 

Lactometer  reading  r 

Total  solids  =  -  --  -f  1.2  fat  +  0.14. 

4 

With  samples  which  do  not  differ  greatly  from  the  average  com- 
position the  results  thus  calculated  are  usually  accurate  within  0.25 
per  cent. 

DETERMINATION  OF  FAT,   PROTEIDS,  MILK  SUGAR,  AND  ASH. 

TOTAL  SOLIDS  AND  ASH. 

Into  an  accurately  weighed  flat-bottomed  platinum  dish  intro- 
duce two  to  five  grams  (depending  upon  the  size  of  the  dish ;  see 

*  Testing  Milk  and  Its  Products.     Madison,  Wisconsin,  1899. 
f  Allen:    Commercial  Organic  Analysis,  Vol.  IV.,  p.  166. 


MILK.  221 

below)  of  the  thoroughly  mixed  milk  and  weigh  quickly  to  the 
nearest  milligram.  If  this  weighing  cannot  be  accomplished  within 
one  minute  the  dish  should  be  covered  with  a  weighed  watch  glass 
to  retard  evaporation.  Place  the  open  dish  on  a  water  bath  or  on 
top  of  the  boiling  water  oven  until  nearly  all  of  the  water  is  ex- 
pelled ;  dry  to  constant  weight  in  a  boiling  water  oven  or  an  air 
bath  kept  constantly  at  97°  to  100°.  The  residue  is  somewhat 
hydroscopic  and  must  be  weighed  quickly  upon  removal  from  the 
desiccator  in  order  to  obtain  the  correct  amount  of  total  solids. 

Ignite  the  dry  solids  in  a  muffle  at  550°  to  600°,  or,  if  this  is  not 
feasible,  regulate  a  Bunsen  burner  to  give  a  very  small  colorless 
flame  and  apply  this  carefully  with  constant  attention  so  that  no 
part  of  the  dish  is  heated  above  the  lowest  possible  redness.  The 
ash  should  be  white  or  very  light  gray.  After  obtaining  the  weight 
of  the  ash  it  may  be  used  in  testing  for  preservatives,  as  described 
below. 

Notes  on  Total  Solids.  —  When  the  same  portion  is  not  to  be  used 
for  the  determination  of  ash  a  platinum  dish  is  not  essential. 
Lead  foil  bottle  caps  are  then  very  convenient,  as  they  are  easily 
numbered  by  scratching,  quickly  heated  and  cooled,  and  so  cheap 
that  each  dish  can  be  rejected  after  being  used  once.  In  order 
that  a  larger  surface  may  be  exposed,  the  dish  may  contain  dry 
sand  or  other  porous  material  and  a  small  stirring  rod,  weighed 
with  the  dish  and  used  to  stir  the  residue  while  drying.  Unless 
absorbed  upon  porous  material,  no  more  than  0.5  gram  of  milk  for 
each  square  centimeter  of  the  area  of  the  bottom  of  the  dish  should 
be  taken  for  the  determination. 

The  methods  used  in  the  Government  Laboratory,  London,  for 
the  determination  of  solids  in  sour  or  fermented  milk  and  the  esti- 
mation of  the  solids  lost  in  fermentation  have  recently  been 
described  by  Thorpe.* 

Notes  on  Ash.  —  Normally  about  two-thirds  of  the  ash  of  cows' 
milk  is  insoluble  in  hot  water.  The  presence  of  a  larger  propor- 
tion of  soluble  ash  may  be  due  to  the  use  of  mineral  preservatives 
or  to  the  addition  of  salts  to  restore  the  density  and  ash  content  to 
milk  which  has  been  watered.  Special  tests  for  some  of  the  mineral 
preservatives  are  given  beyond.  In  important  cases  it  may  be 
necessary  to  analyze  the  ash  to  show  whether  it  is  of  normal  char- 

*  Journ.  Chem.  Soc.,  1905,  87,  206. 


222  ORGANIC   ANALYSIS. 

acter.  Richmond  *  gives  the  following  as  the  average  composition 
of  milk  ash:  Calcium  oxide,  20.27  per  cent.;  magnesium  oxide, 
2.80  per  cent.;  potassium  oxide,  28.71  per  cent;  sodium  oxide,  6.67 
per  cent.;  phosphoric  anhydride,  29.33  percent.;  chlorine,  14.00  per 
cent.;  carbonic  anhydride,  0.97  per  cent.;  sulphuric  anhydride,  trace ; 
ferric  oxide,  etc.,  0.40  per  cent.  According  to  most  other  writers  f 
milk  ash  contains  sulphates,  but  only  traces  of  carbonates.  A 
sample  of  ash  from  the  mixed  milk  of  about  500  cows  which  has 
been  partially  examined  by  Thompson  and  the  writer,  shows  no 
appreciable  amount  of  carbonates  and  only  traces  of  sulphates. 
The  ash  was  prepared  at  known  temperatures  with  the  precautions 
given  in  Chapter  I.  According  to  the  results  thus  far  obtained 
there  was  no  volatilization  of  chlorides  below  650°,  but  even  at  450° 
to  500°  there  was  considerable  loss  of  chlorine  due  doubtless  to 
the  formation  of  acid  products  in  the  combustion  of  the  organic 
constituents  of  the  milk. 

FAT.  —  GRAVIMETRIC  DETERMINATION. 
Adams    Paper  Coil  Method. 

In  this  method  the  milk  is  dried  on  porous  paper,  the  fat  ex- 
tracted by  means  of  ether  into  a  weighed  flask,  the  ether  evapor- 
ated, and  the  fat  weighed. 

Apparatus.  —  (i)  Strips  of  thick  absorbent  fat-free  paper  about 
55  cm.  long  and  6.25  cm.  wide,J  each  rolled  into  a  loose  coil  and 
fastened  by  means  of  a  piece  of  wire  or  fat-free  thread.  If  diffi- 
culty is  found  in  making  a  loose  coil,  two  pieces  of  fat- free  string 
may  be  laid  lengthwise  upon  the  paper  strip  before  rolling  it  up. 
This,  however,  should  not  be  necessary. 

(2)  A  Soxhlet  apparatus  for  ether  extraction;  the  form  having 
ground  glass  connections  being  recommended. 

(3)  A  safety  water  bath  or,  preferably,  an  electric  heater  which 
can  be  easily  regulated. 

Determination.  —  Mix  the  milk  thoroughly  and  absorb  a  known 
amount,  about  5  grams,  on  the  paper  coil.  The  milk  can  be  meas- 
ured by  means  of  a  5  c.c.  pipette  and  delivered  directly  upon  the 

*  Dairy  Chemistry,  p.  32. 

f  See  tabulated  analyses  in  Stohmann's  Milch-  und  Molkereiproducte,  p.  89. 

J  Strips  of  paper  especially  prepared  for  this  purpose  are  made  by  Schleicher  and 
Schiill.  If  these  are  not  available  the  paper  strips  must  be  very  carefully  extracted  be- 
fore use.  See  Richmond's  Dairy  Chemistry,  pp.  91-93. 


MILK.  223 

coil,  but  as  milk  is  more  viscous  than  water  an  ordinary  5  c.c. 
pipette  will  deliver  less  than  5  c.c.  of  milk,  so  that  this  method  can 
be  made  accurate  only  by  determining  experimentally  the  amount 
of  milk  which  the  pipette  actually  delivers.  A  better  method  is  to 
pour  about  5  c.c.  into  a  very  small  beaker,  weigh  quickly  to  centi- 
grams and  at  once  absorb  the  milk  by  standing  the  coil  in  the 
beaker.  The  absorption  can  be  hastened  by  inclining  the  beaker 
and  rotating  the  coil.  The  last  drops  in  the  beaker  must  be 
carefully  absorbed.  Stand  the  coil  upon  the  dry  end  and  re-weigh 
the  beaker  quickly  to  centigrams.  If  carried  out  rapidly  this 
method  is  considerably  more  accurate  than  measuring  with  a 
pipette.  Dry  the  coil  thoroughly  in  a  boiling  water  oven,  place  in 
a  Soxhlet  extractor,  and  extract  with  anhydrous  ether,  using  the 
electric  heater  or  safety  water  bath  and  keeping  the  apparatus  as 
far  as  possible  from  free  flames.  If  not  more  than  5  grams  of  milk 
is  used  and  the  extractor  siphons  at  intervals  of  10  to  15  minutes, 
the  extraction  need  not  be  continued  longer  than  three  hours.  At 
the  end  of  the  extraction  disconnect  the  apparatus,  remove  the 
coil,  replace  the  extractor,  recover  nearly  all  the  ether  by  allowing 
it  to  collect  in  the  space  formerly  occupied  by  the  coil,  return  the 
ether  to  its  bottle,  and  heat  the  flask  containing  the  fat  in  a  boiling 
water  oven  until  the  weight  is  practically  constant. 

Notes.  —  As  the  milk  is  absorbed  by  the  paper  the  greater  part 
of  the  fat  is  left  on  or  near  the  surface,  so  that  it  is  very  rapidly 
extracted  by  the  ether.  The  coil  must  be  thoroughly  dried  before 
extracting  with  ether.  The  drying  can  be  hastened  by  pressing  in 
the  dry  end  of  the  coil  so  that  the  inner  layers  of  the  wet  end  are 
made  to  project  in  the  form  of  a  cone.  Such  a  coil  will  usually  be 
dry  after  standing  two  to  three  hours  in  the  boiling  water  oven. 
On  removing  from  the  oven  press  back  the  projecting  end  of  the 
coil  and  place  it,  milk  end  down,  in  the  extractor;  connect  with 
the  flask,  pour  in  ether  until  it  siphons  into  the  flask,  then  enough 
more  ether  to  cover  about  half  of  the  coil.  This  is  sufficient  to 
avoid  any  danger  of  the  flask  going  dry  during  the  extraction,  if 
the  heat  is  so  regulated  that  no  perceptible  amount  of  ether  escapes 
through  the  reflux  condenser.  To  dry  the  extract  leave  it  in  the 
boiling  water  oven  for  three  hours,  allow  to  cool  for  one-half  hour, 
weigh,  and  then  repeat,  heating  about  one  hour  each  time,  until 
two  successive  weighings  show  a  loss  of  less  than  one  milligram. 
In  laboratories  where  many  determinations  are  made  it  is  cus- 


224  ORGANIC   ANALYSIS. 

ternary  to  dry  the  extract  for  a  fixed  length  of  time  (usually  five 
hours),  which  has  been  found  by  experience  to  be  sufficient. 

Babcock  Asbestos  Method* 

Provide  a  hollow  cylinder  of  perforated  sheet  metal,  60  mm.  long 
and  20  mm.  in  diameter,  closed  5  mm.  from  one  end  by  a  disk  of 
the  same  material.  The  perforations  should  be  about  0.7  mm.  in 
diameter  and  about  0.7  mm.  apart.  Fill  loosely  with  from  1.5  to 
2.5  grams  of  freshly  ignited,  woolly  asbestos,  free  from  fine  and 
brittle  material,  cool  in  a  desiccator,  and  weigh.  Introduce  a 
weighed  quantity  of  milk  (between  3  and  5  grams)  and  dry  at  100° 
to  constant  weight.  This  weight  shows  the  percentage  of  total 
solids.  Place  the  cylinder  in  an  extractor  and  complete  the 
determination  of  fat  as  described  above. 

This  method  avoids  the  possibility  of  having  any  ether-soluble 
matter  in  the  porous  substance  used  to  absorb  the  milk.  It  is 
especially  recommended  for  the  determination  of  fat  in  cream 
which  cannot  be  absorbed  upon  the  paper  coil  without  previous 
dilution. 

PROTEIDS. 

Formerly  milk  proteids  were  precipitated,  or  the  milk  evaporated 
to  dryness,  and  the  residue  after  washing  with  ether  and  dilute 
alcohol  was  dried,  weighed,  burned,  and  the  ash  deducted.  On 
account  of  the  difficulty  of  completely  removing  the  sugar  and  fat, 
the  results  thus  obtained  were  usually  too  high ;  so  that  in  the 
older  statements  of  the  composition  of  milk  (some  of  which  are 
stiil  often  quoted)  the  proteids  were  usually  overestimated. 

Protein  in  milk  is  now  calculated  from  the  nitrogen  content 
multiplying  the  latter  by  the  usual  factor  6.25  or  sometimes  by  a 
special  factor,  6.33  or  6.37,  based  on  analyses  of  milk  proteids 
showing  less  than  16  per  cent,  of  nitrogen. 

To  determine  the  total  nitrogen  in  milk  pour  5  to  10  grams 
of  the  sample  into  a  small  beaker,  weigh  quickly  to  centigrams, 
pour  the  milk  carefully  into  a  Kjeldahl  flask,  re-weigh  the  beaker, 
and  introduce  20  to  25  c.c.  of  concentrated  sulphuric  acid  into  the 
flask  in  such  a  way  as  to  wash  down  any  milk  which  may  have 
remained  in  the  neck.  Add  0.7  gram  of  mercury,  heat  gently  over  a 
very  small  flame  until  most  of  the  water  is  expelled  and  no  more 
frothing  or  spirting  occurs,  then  increase  the  size  of  the  flame  and 
complete  the  determination  as  described  in  Chapter  II. 

*  Bui.  46,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


MILK.  225 

Casein  can  be  precipitated  by  acidulating  the  milk  or  by  means 
of  magnesium  sulphate.  Determination  of  nitrogen  in  the  washed 
precipitate  shows  the  amount  of  casein  in  the  milk.  Albumin  can 
be  precipitated  by  boiling  the  filtrate  and  determined  in  the  same 
manner.  Detailed  directions  for  these  determinations  will  be  found 
in  Bulletin  46,  Bureau  of  Chemistry,  U.  S.  Dept.  Agriculture. 

MILK  SUGAR  OR  LACTOSE. 

In  most  cases  the  direct  determination  of  lactose  is  unnecessary 
as  the  difference  between  the  percentage  of  total  solids  and  the 
sum  of  the  percentages  of  fat,  proteids,  and  ash  should  not  differ 
from  the  true  percentage  of  lactose  by  more  than  O.I  to  O.2  per 
cent.  When  direct  determination  is  desired  either  the  polariscopic 
method  or  one  of  the  methods  based  upon  the  reduction  of  copper 
can  be  used.  In  the  former  case  the  proteids  are  precipitated  and 
the  solution  clarified  by  means  of  mercuric  nitrate  or  iodide,  in  the 
latter  by  cupric  hydroxide  or  acetic  acid,  alum,  and  aluminum 
hydroxide. 

Optical  Determination* 

Place  65.8  grams  of  milk  in  each  of  two  flasks,  one  graduated 
at  100,  the  other  at  200  c.c.,  to  each  add  4  c.c.  of  mercuric  nitrate 
solution,!  fill  to  the  mark,  shake,  filter  through  dry  paper,  and 
polarize  in  a  200  mm.  tube  in  the  Schmidt  and  Haensch  polariscope. 

In  each  case  the  reading  is  too  high  on  account  of  the  volume 
occupied  by  the  precipitate  which  contains  the  proteids  and  fat  of 
the  milk.  This  volume  is  twice  as  great  in  proportion  in  the  100 
c.c.  as  in  the  200  c.c.  flask.  The  corrected  reading  and  the  volume 
occupied  by  the  precipitate  can,  therefore,  be  calculated  by  the 
method  of  double  dilution  as  in  the  following  example:  —  J 

Weight  of  milk  taken  65 .8  grams,  or  twice  the  "  lactose  normal  " 
weight  §  for  the  Ventzke  scale. 

*  Wiley  and  Ewell :  Journ.  Amer.  Chem.  Soc.,  1896,  18,  428. 

f  To  prepare  this  solution  dissolve  mercury  in  twice  its  volume  of  nitric  acid,  1.42 
specific  gravity,  and  add  to  the  solution  an  equal  volume  of  water  ;  or  prepare  a  solu- 
tion of  equal  strength  by  dissolving  solid  mercuric  nitrate  in  water  acidulated  with 
nitric  acid. 

£  Compare  Wiley's  Agricultural  Analysis,  Vol.  III.,  pp.  102,  278. 

§  Calculated  from  the  sucrose  normal  weight  and  the  approximate  specific  rotatory 
powers  of  sucrose  and  lactose.  These  data  and  directions  for  the  manipulation  of  the 
polariscope  have  been  given  in  Chapters  V.  and  VI. 


226  ORGANIC   ANALYSIS. 

Average  reading  from  100  c.c.  flask,  10.45. 

"  "  "      200  c.c.     "         5-0/5. 

Then 

10.45  —  (5-0/5  x  2)  =  0.30  (half  the  error  in  the  higher  reading). 

10.45  —  (°-30  x  2)    =  9.85  (corrected  reading  for  100  c.c.  flask). 

9.85  -7-  2  =  4.925,  corrected  percentage  of  lactose. 

The  volume  of  the  precipitate  is  calculated  as  follows : 
10.45  -s-  2  =  5.225,  apparent  percentage  of  lactose  (100  c.c.  flask). 

Then 

5.225  :  4.925  ::  100:  x. 

x=  94.26,  the  volume  of  solution  in  the  100  c.c.  flask.  Hence 
the  volume  of  the  precipitate  is  5.74  c.c. 

Determination  by  Fehling  Solution* 

Dilute  25  c.c.  of  milk  with  400  c.c.  of  water  in  a  500  c.c.  flask, 
add  10  c.c.  of  the  copper  sulphate  solution  used  in  the  Fehling 
method,  mix  and  add  4.4  c.c.  of  normal  sodium  or  potassium  hy- 
droxide (or  an  equivalent  amount  of  a  weaker  standard  solution), 
fill  to  the  mark,  mix,  and  filter  through  dry  paper.  The  filtrate 
must  contain  copper  in  order  to  ensure  the  absence  of  any  trace  of 
free  alkali.  In  this  clear  filtrate  lactose  can  be  determined  by 
means  of  Fehling  solution  either  by  Defren's  method  as  described 
in  Chapter  V.,  or  by  Soxhlet's  method,  Bui.  46,  /.  c.  The  milk  is  so 
greatly  diluted  in  clarifying  the  solution  that  the  volume  of  the 
precipitated  proteids  and  fat  can  be  neglected. 

If  lactose  is  to  be  determined  volumetrically,  the  proteids  can  be 
precipitated  and  the  solution  clarified  as  described  in  Richards 
and  Woodman's  Air,  Water,  and  Food. 

INTERPRETATION  OF  RESULTS. 

The  principal  adulterations  affecting  the  percentages  of  nutri- 
ents in  milk  are  the  addition  of  water  (sometimes  containing  dis- 
solved solids)  and  the  removal  of  cream.  These  adulterations  are 
sometimes  difficult  to  detect  with  certainty  because  genuine  cows' 
milk  varies  considerably  both  in  fat  and  in  other  solids.  Since  the 
percentage  of  fat  is  more  variable  than  that  of  solids  not  fat,  skim- 
ming is  more  difficult  to  detect  than  watering.  If  as  much  as  one- 
fourth  of  the  fat  were  removed,  the  skimming  would  usually  be  in- 

*Bul.  46,  loc.  cit. 


MILK. 


227 


dicated  by  the  disturbance  of  the  normal  relation  between  the 
percentage  of  fat  and  that  of  proteids  or  of  solids  not  fat,  but  the 
analysis  cannot  be  said  to  prove  the  removal  of  cream  unless  it 
shows  a  lower  percentage  of  fat  than  is  ever  found  in  genuine  nor- 
mal milk.  Starting  with  average  milk  containing  4  per  cent,  fat 
and  9  per  cent,  solids  not  fat,  one-tenth  of  the  fat  could  be  re- 
moved by  skimming  and  the  resulting  product  containing  3.6  per 
cent,  fat  could  not  be  distinguished  by  analysis  from  genuine  milk, 
while  if  the  fat  were  reduced  to  3.6  per  cent,  by  watering,  the 
solids  not  fat  would  be  reduced  to  8.1  per  cent,  which  is  sufficiently 
below  the  normal  to  be  detected  without  difficulty.  Occasionally 
genuine  milk  contains  even  less  than  8.0  per  cent,  of  solids  not 
fat  (the  deficiency  in  most  of  these  cases  falling  mainly  upon  the 
milk  sugar)  so  that  the  limit  of  8.5  for  solids  not  fat  might  indi- 
cate watering  where  none  had  been  practiced.  Such  errors  are 
avoided  by  taking  account  of  the  proteids  and  ash.  Milk  should 
contain  not  less  than  8.5  per  cent,  of  solids  not  fat,  3.0  per  cent, 
of  proteids,  0.7  per  cent,  of  total  ash,  0.5  per  cent,  of  ash  insoluble 
in  hot  water.  These  four  determinations  will  usually  suffice  to 
show  whether  the  milk  is  genuine  or  has  been  watered  with  or 
without  the  addition  of  soluble  solids. 

UNITED  STATES  AND  STATE  STANDARDS  FOR  MILK,  1905. 


Total 
Solids, 
Per  Cent. 

325 
&** 

Fat, 
Per  Cent. 

Ill 
H-l 

Solids 
not  Fa', 
Per  Cent. 

Fat, 
Per  Cent. 

U.  S.  Dept.  Agric.. 

12 

8.5 

3.25 

New  York  

12 

7 

District  of  Columbia 

•j  c 

North  Carolina 

12 

8  5 

•i  25 

Georgia 

8  5 

3  5 

North  Dakota 

12 

7 

8J 

7, 

Ohio 

12 

7 

Illinois  

7 

"     May—  Tune 

lie 

Indiana  



7. 

Oregon          

12 

7 

Iowa  

12.5 

7 

i  Pennsylvania 

12 

7 

Kentucky  

12 

7 

Porto  Rico  

12 

7 

Maine  . 

12 

•2 

Rhode  Island 

12 

2  5 

Maryland 

12.5 

1C 

South  Carolina 

8.5 

7 

Massachusetts  . 

I-J 

99 

7.7 

South  Dakota 

IT. 

7 

"     Apr.  -Sept. 
Michigan  

12 

12.5 

9 

3 

1 

Utah  
Vermont.. 

12.5 

12.  5 

0.25 

3 

Minnesota  

17 

7.5 

'  '     May-June. 

12 

Montana  

J 
ii.  5 

7 

Washington  

8 

7 

New  Hampshire. 

13 

0  5 

7  e 

\Visconsin 

1 

"     Apr.-Sept. 

T7 

7 

Wyoming 

12 

2.4. 

New  Jersey  

12 

"     May-June.. 

"•5 



In  most  cases  it  is  not  necessary  to  show  conclusively  whether 
milk  has  been  skimmed  or  watered,  but  only  whether  it  meets  the 


228  ORGANIC   ANALYSIS. 

requirements  of  a  legal  or  trade  standard.  The  principal  standards 
in  force  in  the  United  States  on  April  I,  1905,  are  given  in  the  ac- 
companying table  (p.  227)  from  Circular  No.  74,  Bureau  of  Animal 
Industry,  U.  S.  Dept.  Agriculture. 

PRESERVATIVES   AND    PRODUCTS    OF    FERMENTATION. 
Only  a  few  of  the  many  products  produced  by  the  action  of 
micro-organisms  in  milk  and  of  the  substances  used  to  retard  the 
formation  of  such  products  or  to  hide  their  presence  can  be  con- 
sidered in  this  chapter.* 

FORMALDEHYDE. 
Detection. 

Of  the  many  methods  available  for  the  detection  of  formalde- 
hyde in  milk  only  three  of  the  best  known  and  most  delicate  will 
be  given. 

Sulphuric  Acid  Test.\ — Dilute  2  to  3  c.c.  of  milk  with  an  equal 
volume  of  water  in  a  test  tube,  add  carefully  so  as  not  to  mix  the 
layers  from  3  to  5  c.c.  of  concentrated  commercial  sulphuric  acid 
or  pure  acid  to  which  a  trace  of  ferric  salt  has  been  added.  If 
formaldehyde  is  present  a  violet  ring  forms  at  the  junction  of  the 
two  liquids.  The  charring  of  the  milk  by  the  sulphuric  acid, 
makes  it  difficult  to  define  the  delicacy  of  the  test.  One  part  of 
formaldehyde  in  100,000  of  milk  can  be  detected  if  the  milk  is 
fresh  and  the  test  is  applied  soon  after  adding  the  preservative. 

Hydrochloric  Acid  Test.\ — Mix  10  c.c.  of  milk  and  10  c.c.  of  con- 
centrated hydrochloric  acid  containing  about  2  mgs.  of  ferric 
chloride  and  heat  slowly  nearly  to  boiling,  rotating  the  mixture 
occasionally  to  ensure  solution  of  the  curd.  In  the  presence  of 
formaldehyde  a  violet  color  develops,  otherwise  the  solution 
slowly  turns  brown.  The  test  is  best  performed  in  a  porcelain 
casserole  and  in  case  of  doubt  the  violet  color  is  made  much  more 
distinct  by  adding  50  to  75  c.c.  of  water  after  having  heated  just 
below  boiling  for  about  a  minute.  The  liquid  must  be  observed 

*  Trade  names  and  analyses  of  many  proprietary  preservatives  will  be  found  in  the 
Year-Book  of  the  U.  S.  Department  of  Agriculture  for  1900,  Chapin's  Theory  and 
Practice  of  Infant  Feeding,  and  the  Zeitschrift  fur  die  Untersuchung  der  Nahrungs- 
und  Genussmittel. 

f  Hehner  :  Analyst,  1896,  21,  95. 

%  Leach:  Ann.  Rpts.  Mass.  State  Board  of  Health,  1897,  558  ;  1899,  699  ;  Food 
Inspection  and  Analysis,  p.  140.  See  also  Chapter  IV. 


MILK.  229 

carefully  at  the  moment  of  dilution  as  the  color  which  is  brought 
out  in  this  way  fades  very  rapidly.  This  test  is  delicate  to  I  :  250,- 
ooo  but  formaldehyde  added  to  milk  in  such  small  quantities  soon 
disappears.  When  added  to  the  extent  of  I  :  50,000  to  I  :  100,000 
the  presence  of  formaldehyde  in  the  milk  will  be  shown  by  this 
test  for  from  I  to  5  days.*  In  testing  sour  or  stale  milk  the 
brown  color  noted  above  will  often  obscure  the  reaction  given  by 
a  small  amount  of  formaldehyde  until  the  solution  is  diluted  with 
water,  but  at  this  point  the  violet  color  can  be  seen  even  though 
the  milk  may  have  been  much  charred  by  the  acid. 

Gallic  Acid  Test.  —  This  test  has  been  described  in  Chapter  V. 
To  apply  it  to  milk,  acidulate  30  c.c.  with  2  c.c.  of  normal  sul- 
phuric acid  and  distil.  To  the  first  5  c.c.  of  distillate  add  0.2  to 
0.3  c.c.  of  a  saturated  solution  of  gallic  acid  in  pure  alcohol,  incline 
the  test  tube  and  pour  in  slowly  3  to  5  c.c.  of  concentrated  sul- 
phuric acid.  The  presence  of  formaldehyde  is  shown  by  the 
characteristic  blue  ring  described  in  detail  in  Chapter  V.  In  the 
writer's  experience  this  test  is  at  least  twice  as  delicate  as  either 
the  sulphuric  or  the  hydrochloric  acid  test.  The  latter  would  be 
sufficiently  delicate  for  all  practical  purposes  if  milk  samples  could 
always  be  tested  while  fresh,  but  when  small  amounts  of  formalde- 
hyde have  been  added  one  or  two  days  previously,  the  gallic  acid 
test  may  show  the  preservative  where  either  of  the  other  tests 
would  fail.  This  gallic  acid  reaction  may  also  be  used  very  satis- 
factorily as  a  means  of  confirming  any  doubtful  results  obtained 
by  either  of  the  preceding  tests  as  there  is  little  danger  of  inter- 
ference due  to  charring  or  to  the  appearance  of  other  colors. 

Determination. 

Very  small  amounts  of  formaldehyde  in  milk  can  be  determined 
by  the  following  method,  which  is  essentially  that  of  Smith,f  except 
that  a  larger  quantity  of  sample  is  used. 

To  300  c.c.  of  milk  in  a  round-bottomed  flask  of  about  one  liter 
capacity  add  3  c.c.  of  (I  :  3)  sulphuric  acid  and  some  glass  beads  to 
prevent  bumping,  heat  gradually  to  boiling,  preferably  by  means 
of  a  small  rose-top  burner,  and  distil  until  the  distillate  measures 
60  c.c.  Transfer  this  to  a  ioo  c.c.  flask,  add  10  c.c.  of  standard 

*  Rivas  :    University  of  Pennsylvania  Medical  Bulletin,   1904,17,   175.     Unpub-X 
lished  experiments  by  Williams  and  the  writer. 
•\Journ.  Amer.  Ghent.  Sot:.,  1903,  25,  1036. 


230  ORGANIC   ANALYSIS. 

potassium  cyanide  solution,  approximately  tenth-normal,  and  mix; 
add  a  mixture  of  15  c.c.  of  tenth-normal  silver  nitrate  and  6  to  8 
drops  of  50  per  cent,  nitric  acid,  fill  to  the  mark,  shake,  and  filter 
through  dry  paper.  Determine  the  excess  of  silver  by  the  Volhard 
method  and  calculate  the  results  as  explained  in  Chapter  V.  under 
the  description  of  the  cyanide  method  as  used  for  commercial 
solutions  of  formaldehyde.  The  precautions  and  the  directions 
for  standardizing  there  given  should  also  be  noted. 

By  this  method  from  32  to  39  per  cent,  of  the  formaldehyde  in 
the  milk  is  recovered  and  determined  in  the  distillate.  Assuming 
that  the  amount  recovered  represents  35  per  cent,  of  the  quantity 
in  the  milk,  the  latter  can  be  estimated  with  a  probable  error 
of  about  one-tenth.  Using  this  as  a  means  of  studying  the  disap- 
pearance of  formaldehyde  in  milk,  it  was  found  in  a  typical  experi- 
ment in  which  the  proportion  added  was  I  :  40,000,  that  nearly 
three-fourths  of  the  preservative  had  disappeared  after  two  days  at 
room  temperature.  After  four  days  no  formaldehyde  was  shown 
by  this  method  but  the  violet  color  on  dilution  with  water  after 
heating  with  hydrochloric  acid  containing  ferric  chloride  was  un- 
mistakable. The  latter  reaction  could  still  be  obtained  after  the 
mixture  had  stood  for  two  weeks.  When  formaldehyde  is  added 
to  milk  in  large  proportion,  I  :  1000  to  I  :  10,000  as  in  the  preserva- 
tion of  samples  for  analysis  or  reference,  the  rate  of  disappearance 
is  much  slower. 

HYDROGEN  PEROXIDE. 
Detection. 

Hydrogen  peroxide  in  uncooked  milk  is  easily  detected  by  add- 
ing to  10  to  15  c.c.  of  the  milk,  2  to  3  drops  of  a  2  per  cent,  aque- 
ous solution  of  paraphenylenediamine  hydrochloride.  In  the 
presence  of  hydrogen  peroxide  a  blue  color  appears  either  imme- 
diately upon  shaking  or  after  a  few  minutes  depending  upon  the 
amount  present.  The  reaction  depends  upon  the  action  of  an 
oxidizing  enzyme  in  the  milk  and  the  condition  of  the  milk,  there- 
fore, affects  the  delicacy  of  the  test.  According  to  Arnold  and 
Mentzel  *  I  part  in  40,000  can  be  detected.  Under  ordinary  con- 
ditions the  delicacy  is  probably  somewhat  less  than  this.  In  com- 
parative tests  made  immediately  after  adding  the  same  amounts  of 

*  Ztschr.  Unters.  Nahr.-Genussm.,  1903,  6,  306. 


MILK.  231 

peroxide  to  sweet  milks  I  to  2  days  old  and  to  very  sour  curdled 
milks  3  to  4  days  old,  the  former  were  found  to  give  the  reaction 
much  more  strongly  than  the  latter.  In  practice,  however,  the 
preservative  would  be  added  while  the  milk  was  sweet  and  would 
probably  disappear  entirely  before  the  occurrence  of  curdling. 
Milk  which  has  been  boiled  can  be  tested  after  adding  an  equal 
volume  of  fresh  milk  known  to  be  free  from  peroxide. 

Determination. 

Chick,*  in  an  investigation  of  the  germicidal  properties  and  rate 
of  disappearance  of  hydrogen  peroxide  in  milk,  used  a  method 
based  upon  the  titration  of  the  iodine  liberated  by  the  peroxide  on 
adding  potassium  iodide  and  sulphuric  acid. 

2KI  +  H202  +  H2S04  =  K2S04  +  I2  +  H20. 

Mettlerf  has  used  the  method  with  satisfactory  results  in  the 
following  modified  form  :  To  40  c.c.  of  water,  0.5  gram  of  potas- 
sium iodide,  and  10  c.c.  of  12  per  cent,  sulphuric  acid  in  a  glass- 
stoppered  flask,  add  10  c.c  of  the  milk,  stopper  tight  and  allow  to 
stand  in  a  cool  dark  place  for  two  and  one-half  hours.  In  order  to 
guard  against  any  possible  loss  of  iodine  during  this  time,  use  a  flask 
with  flaring  mouth  as  described  in  connection  with  the  determina- 
tion of  the  iodine  number  (Chapter  IX.),  and  fill  the  gutter  around 
the  stopper  with  a  solution  of  potassium  iodide.  Finally  titrate 
the  iodine  which  has  has  been  set  free  in  the  milk,  using  a  fiftieth- 
normal  solution  of  sodium  thiosulphate.  In  this  titration  it  is  not 
necessary  to  use  starch  as  indicator  since  the  disappearance  of  the 
yellow  color  produced  by  the  action  of  the  iodine  upon  the  pro- 
teids  affords  a  satisfactory  end  point.  Test  analyses  gave  results 
about  3  per  cent,  too  low,  doubtless  because  of  the  absorption  of 
iodine  by  the  milk  fat.  This  source  of  error  can  be  avoided  by 
curdling  the  milk  with  acid,  filtering,  and  adding  the  iodide  to  a 
measured  amount  of  filtrate  ;  but  this  is  considered  inadvisable  in 
view  of  the  fact  that  the  peroxide  may  be  undergoing  decomposi- 
tion during  the  filtration. 

SALICYLIC  ACID  AND  SALICYLATES. 

Harm,}  in  experiments  upon  some  of  the  methods  most  com- 
monly recommended  for  the  extraction  of  salicylic  acid  from  foods, 

*  Centralblatl  fur  Bacteriologie  und  Parasitenkunde,  II.  Abth.,  1901,  I,  705. 
f  Thesis  for  the  degree  of  Bachelor  of  Science,  Columbia  University,  1905. 
£  Thesis  for  the  degree  of  Bachelor  of  Science,  Columbia  University,  1905. 


232  ORGANIC   ANALYSIS. 

recovered  only  a  small  percentage  of  the  amount  known  to  be 
present  in  milk.  Separation  of  salicylic  acid  by  distillation  with 
steam  is  extremely  slow,  and  extraction  by  shaking  with  ether  or 
chloroform  after  precipitating  the  proteids  by  mercuric  nitrate,  is 
usually  very  incomplete.  Better  results  can  be  obtained  by  the 
following  modification  of  Remont's  method:  Acidulate  20  c.c.  of 
milk  with  sulphuric  acid,  shaking  well  to  break  up  the  curd  ;  add 
25  c.c.  of  ether,  mix,  and  allow  to  stand.  Draw  off  5  c.c.  of  the 
ether  layer,  evaporate  to  dryness,  boil  the  residue  with  10  c.c.  of  40 
per  cent,  alcohol,  cool,  adjust  the  volume  to  10  c.c.,  filter  through 
dry  paper  if  necessary  to  remove  fat,  and  test  5  c.c.  of  the  filtrate 
(representing  2  c.c.  of  milk)  by  adding  2  c.c.  of  2  per  cent,  solution 
of  ferric  chloride.  A  purple  or  violet  color  is  produced  if  salicylic 
acid  is  present,  and  approximately  quantitative  results  can  be  ob- 
tained by  diluting  to  50  c.c.  and  matching  with  standard  solutions, 
observing  all  the  precautions  ordinarily  required  in  colorimetric 
work. 

BORIC  ACID  AND  BORATES. 

In  routine  work  *  the  ash  obtained  in  the  usual  course  of  analysis 
is  treated  with  two  drops  of  dilute  hydrochloric  acid  f  and  about 
a  cubic  centimeter  of  water.  A  strip  of  turmeric  paper  is  then 
placed  in  the  dish,  allowed  to  soak  for  a  minute,  removed  and 
allowed  to  dry  in  the  air.  A  deep  red  color  changing  to  green  or 
blue  when  treated  with  dilute  alkali  shows  the  presence  of  boric 
acid.  According  to  Leach  this  reaction  is  delicate  to  I  part  in 
8000.  The  well-known  flame  test  with  methyl  alcohol  is  less  deli- 
cate but  can  be  used  in  confirmation. 

The  turmeric  test  is  very  satisfactory  for  the  detection  of  boric 
acid  in  the  ash,  but  since  a  considerable  loss  of  boron  may  occur 
in  burning  off  the  organic  solids  of  milk,  it  is  better  in  all  impor- 
tant cases  to  take  a  separate  portion  for  this  test  and  make  it 
strongly  alkaline  with  sodium  or  calcium  hydroxide  before  evapo- 
rating to  dryness.  All  of  the  boric  acid  then  remains  in  the  ash 
and  can  be  determined  quantitatively  by  Thomson's  method. J 

*  Leach  :  Food  Inspection  and  Analysis,  p.  144. 

f  Care  should  be  taken  to  note  whether  any  effervescence  occurs.  See  carbonates 
and  bicarbonates  beyond. 

J  Glasgow  City  Anal.  Soc.  Reports,  1895  ;  Analyst,  1896,  21,  64;  Button's  Volu- 
metric Analysis,  8th  Ed.,  p.  100,  Qth  Ed.,  p.  98  ;  U.  S.  Dept.  Agriculture,  Bur.  Chem., 
Bui.  65,  p.  no.  The  standard  alkali  used  in  the  final  titration  should  be  freshly 
standardized  against  boric  acid. 


MILK.  233 

FLUORIDES. 

If  milk  containing  a  small  amount  of  fluoride  is  evaporated  and 
the  solids  burned  to  ash  in  the  usual  way  the  fluorine  is  likely  to 
be  almost  entirely  lost.  In  the  presence  of  a  considerable  excess 
of  alkali  this  loss  of  fluorine  does  not  occur. 

To  detect  or  determine  fluorides,  add  I  gram  of  sodium  carbo- 
nate to  100  c.c.  of  milk,  evaporate,  and  burn  to  ash.  If  only  quali- 
tative results  are  required,  examine  for  fluorides  by  the  well-known 
etching  test  on  glass.  For  a  quantitative  determination  of  the 
fluorine,  leach  the  mixture  of  ash  and  sodium  carbonate  thoroughly 
with  hot  water,  nearly  neutralize  with  sulphuric  acid,  leaving  the 
solution  slightly  alkaline,  and  then  apply  Rose's  method  as  modi- 
fied by  Treadwell  and  Koch:  Ztschr.  anal.  Chem.,  1904,  43,  469. 

CARBONATES  AND  BICARBONATES. 

When  milk  contains  the  equivalent  of  0.05  per  cent,  of  sodium 
carbonate  the  ash  obtained  by  direct  ignition  of  the  solids  shows 
effervescence  on  addition  of  hydrochloric  acid.  Such  effervescence 
is  rarely  if  ever  seen  in  the  ash  of  pure  milk,  but  since  Richmond 
has  found  small  amounts  of  carbonic  acid  in  the  ash  of  milk  be- 
lieved to  have  been  pure,*  the  presence  of  carbonate  or  bicarbo- 
nate should  be  confirmed  by  applying  Schmidt's  test,  in  which 
10  c.c.  of  milk  are  mixed  with  an  equal  volume  of  alcohol  and  a 
few  drops  of  a  I  per  cent,  solution  of  rosolic  acid.  The  color  is 
brownish  yellow  in  pure  milk  but  rose-red  in  milk  containing  car- 
bonate or  bicarbonate.  A  comparative  test  with  pure  milk  should 
always  be  made.  The  reaction  is  nearly  as  delicate  as  the  test  for 
effervescence  in  the  ash. 

Carbonates  and  bicarbonates  are  not  to  be  regarded  as  preserva- 
tives, but  as  adulterants  added  to  hide  the  presence  of  acid  and 
thus  give  the  milk  a  fraudulent  appearance  of  freshness. 

ACIDITY. 

To  determine  the  total  acidity  of  milk,  dilute  10  c.c.  of  sample 
with  90  c.c.  of  water  and  titrate  with  tenth-normal  sodium  or 
potassium  hydroxide  using  phenolphthalein  as  indicator.  The 
acidity  is  usually  considered  to  be  due  mainly  to  lactic  and  car- 

*  The  use  of  salicylates  or  other  alkali  salts  of  organic  acids  as  preservatives  may 
also  give  rise  to  carbonates  in  the  ash. 


234  ORGANIC  ANALYSIS. 

bonic  acids.  In  order  to  eliminate  the  carbonic  acid,  the  mixture 
of  milk  and  water  may  be  boiled  in  a  beaker  loosely  covered  with 
a  watch  glass  for  ten  minutes.  Both  the  total  acidity  and  the 
acidity  after  boiling  are  often  calculated  as  due  to  lactic  acid.  The 
results  are  also  frequently  given  as  "  degrees  of  acidity."  This 
term  unfortunately  has  no  definite  meaning  when  applied  to  milk 
and  must  be  explained  in  each  case.  It  is,  therefore,  recommended 
that  results  be  expressed  as  "  total  acidity  equivalent  to  per  cent,  of 
lactic  acid  "  and  "  acidity  after  boiling  equivalent  to  per  cent,  of 
lactic  acid." 

In  a  very  recent  paper  *  Thorpe  recommends  that  the  volatile 
acids  be  calculated  as  acetic  acid  and  determined  as  follows :  To 
10  grams  of  milk  in  a  platinum  dish,  add  a  little  phenolphthalein 
and  one-half  the  volume  of  tenth-normal  sodium  hydroxide  which 
would  be  required  to  neutralize  the  total  acidity.  Evaporate  to 
dryness  on  a  water  bath  with  frequent  stirring,  add  20  c.c.  of 
boiling  distilled  water,  stir  thoroughly  to  disintegrate  and  detach 
the  milk  solids  from  the  dish,  and  titrate  with  tenth-normal  sodium 
hydroxide.  The  difference  between  the  total  volume  of  alkali 
used  in  this  determination  and  the  volume  required  to  neutralize 
10  grams  of  the  original  milk  gives  a  measure  of  the  volatile  acids 
in  the  sample. 

The  causes  and  significance  of  the  acidity  of  milk  have  been 
studied  recently  by  Touchat,f  Vieth  and  Siegfeld,|  Richmond  and 
Harrison,  §  Kirsten,  ||  and  Haune.  *f 

AMMONIA. 

The  Boussingault-Shaffer  method  for  the  determination  of  am- 
monia in  urine  **  has  been  thoroughly  studied  by  Berg  ft  w'tn 
regard  to  its  applicability  to  the  much  smaller  amounts  of  ammonia 
which  occur  in  milk.  The  following  is  an  outline  of  the  method  as 
adapted  to  milk  :  — 

*  Journ.    Chem.  Soc.,  1905,  87,  220. 

•\  British  Food  Journ.,  1899,  199;  Abs.  Jour.  Chem.  Soc.,  1900,  78,  ii,  582. 

J  Milch  Ztg.,  29,  593;  Abs.  Jahr.  Thierchem.,  1900,  261. 

\  Analyst,  1900,  25,  I2O. 

||  Ztschr.  Unters.  Nahr.-Genussm.,  1902,  5,  97. 

^  Milch  Ztg.,  1904,   33,   659,  679,  709,  725;  Abs.   Experiment  Station  Record 

1905,  I6>  595- 

**  Shaffer:  Amer.  Journ.  Physiol.,  1903,  8,  330. 
ft  Thesis  for  the  degree  of  Bachelor  of  Science,  Columbia  University,  1904. 


MILK.  235 

Fifty  c.c.  of  the  sample  are  mixed,  in  a  two-liter  round-bottomed 
flask,  with  an  equal  volume  of  methyl  alcohol,  10  grams  of  sodium 
chloride,  and  0.5  gram  of  sodium  carbonate.  The  latter  liberates 
the  ammonia  from  its  salts,  the  salt  diminishes  the  dissociation  of 
the  carbonate  and  thus  prevents  or  retards  the  cleavage  of  am- 
monia from  the  nitrogenous  organic  compounds  in  the  milk,  and 
the  alcohol  also  tends  to  prevent  cleavage  both  by  diminishing 
dissociation  and  by  lowering  the  boiling  point  of  the  mixture. 
The  flask  is  closed  with  a  stopper  having  an  inlet  tube  to  provide 
for  the  admission  of  air  and  an  outlet  fitted  with  a  distilling  head 
and  connected  with  two  condensing  cylinders  (in  series)  containing 
together  from  15  to  25  c.c.  of  twentieth-normal  acid  diluted  with 
water  to  40  c.c.  The  cylinders  are  connected  with  a  suction  pump. 
By  this  arrangement  it  is  possible  to  distil  the  ammonia  into  the  ab- 
sorption cylinders  completely  within  fifteen  minutes  at  a  tempera- 
ture not  above  60°. 

Fresh  milk  of  good  quality  yields  less  than  o.ooi  per  cent,  of 
ammonia  when  treated  by  this  method.  If  the  sodium  chloride  is 
omitted  in  the  distillation  a  larger  amount  of  ammonia  is  obtained. 
For  convenience  the  ammonia  thus  easily  split  off  from  organic 
matter  by  dilute  sodium  carbonate  at  55°  to  60°  is  called  "  cleav- 
age ammonia."  Both  the  ammonia  existing  as  such  in  the  milk 
and  the  "  cleavage  ammonia "  increase  rapidly  under  conditions 
which  favor  the  decomposition  of  proteids  by  bacteria  or  molds, 
and  it  is  believed  that  these  determinations  will  prove  useful  in  the 
sanitary  examination  of  milk.  The  methods  and  results  are  more 
fully  discussed  in  a  paper  on  the  determination  of  ammonia  in 
milk  in  the  Journal  of  the  American  Chemical  Society  for  February, 
1905. 

REFERENCE  BOOKS. 

Allen :  Commercial  Organic  Analysis,  Vol.  IV. 

Babcock  :  Reports  of  Wisconsin  Experiment  Station. 

Blyth  :  Foods,  their  Composition  and  Analysis,  5th  Ed.  London, 
1903. 

Chapin  :  Theory  and  Practice  of  Infant  Feeding,  2d  Ed.  New 
York,  1904. 

Conn :  Bacteria  in  Milk  and  Its  Products.  Phila.,  1903,  and 
Reports  of  Conn.  (Storrs)  Expt.  Station. 

Fleischmann  :  Lehrbuch  der  Milchwirthschaft.     Bremen,  1893. 


236  ORGANIC   ANALYSIS. 

Hammarsten :  Physiological  Che  nistry,  4th  English  Edition  by 
Mandel.  New  York,  1904. 

Konig  :  Die  Menschliche  Nahrungs-  tmd  Genussmittel,  4th  Ed., 
Berlin,  1903. 

Leach:  Food  Inspection  and  Analysis,  New  York,  1904. 

Raudnitz  :  Bestandteile,  Eigenschaften  und  Veranderungen  der 
Milch,  in  Asher's  Ergebnisse  der  Physiologic,  2,  I.  Wiesbaden, 
1903. 

Richmond:  Dairy  Chemistry.     London,  1899. 

Rothschild:  Bibliographia  Lactaria.     Paris,  1901. 

Russell:  Dairy  Bacteriology.     Madison,  Wis.,  1899. 

Stohmann  :  Milch-  und  Molkereiproducte.     Braunschweig,  1898, 

Swithinbank  and  Newman :  Bacteriology  of  Milk.  London, 
1903. 

Wiley  :    Agricultural  Analysis,  Vol.  III. 


ADDITIONAL   NOTES   AND    CORRECTIONS. 

Page  3. — Note  also  Morse  and  Taylor's  new  method  of  ultimate 
analysis:  Amer.  Chem.  Journ.,  1905,  33,  591. 

Page  6,  line  22. — Read  sometimes  instead  of  some. 

Page  10. — Additional  references,  Milbauer :  Quantitative  Stick- 
stoffbestimmung  in  Hydrazonen  und  Osazonen  nach  der  Methode 
von  Kjeldahl,  Ztschr.  Zuckerind,  Bohmen,  28,  338;  Abs.  Biochem. 
Central.,  1905,  469.  Flamand  and  Prager  :  Analyse  von  Verbindun- 
gen  mit  Stickstoff-StickstofT-Bindung  nach  Kjeldahl- Methode,  Ber. 
dent.  chem.  Ges.,  1905,  38,  559. 

Page  1 8. — A  modified  form  of  the  sodium  peroxide  method  is 
recommended  by  Neumann  and  Meinertz:  Ztschr.  physiol.  Chem., 
1904,  43,  37.  See  also  Dubois:  Journ.  Amer.  Chem.  Soc.,  1905,  27, 
729. 

Page  26,  last  paragraph. — Note  :  Addition  of  calcium  carbonate 
suffices  for  most  sweet  wines,  but  samples  containing  much  free 
acid  should  always  be  neutralized  carefully,  using  litmus  paper  as 
indicator. 

Page  35 — Additional  reference  on  determination  of  fusel  oil, 
Schidrowitz:  Analyst,  1905,  30,  190. 

Page  41. — Additional  reference,  Suhr  :  Kritische  Studien  liber  die 
quantitative  Bestimmung  des  Glycerins,  Archiv  fur  Hygiene,  1892, 

4i,  305. 

Page  65,  line  25. — Read  Maquenne  instead  of  Macquenne. 

Page  70. — For  discussion  of  ferrous  thiocyanate  as  indicator  in 
the  volumetric  determination  of  reducing  sugars  see  Ling  and 
Rendle :  Analyst,  1905,30,  182. 

Page  78. — The  details  of  manipulation  and  factors  for  calculation 
of  furfurol  and  pentosans  should  be  modified  according  to  Krober  : 
Journ.  Landw.,  1900,  48,  357;  Ztschr.  angew.  Chem.,  1902,  477; 
U.  S.  Dept.  Agr.,  Bur.  Chem,  Bui.  73,  p.  173. 

Page  in. — Add  to  references  on  detection  of  dextrose,  Wood: 
Chemical  and  Microscopical  Diagnosis,  New  York,  1905. 

Page  164. — Add  to  references  on  detection  of  cottonseed  oil, 
Tolman :  Journ.  Amer.  Chem.  Soc.,  1905,  27,  589. 


237 


SUBJECT   INDEX. 


Absolute  alcohol,  24,  36 

Acetaldehyde,  47,  53,  54,  55,  57 

Acetates,  112 

Acetin,  39 

Acetone,  52,  54,  55,  57 

Acetylation,  39,  127,  138 

Acetyl  number,  127,  138 

Acid,  acetic,  33,  67,  112,  114,  116 

arachidic,  120,  152 

barbituric,  77 

behenic,  120 

boric,  232 

brassidic,   121 

butyric,  112,  115,  119 

capric,  119 

caproic,  114,  119 

caprylic,  114,  119 

carbonic,  68 

carnaiibic,  1 20 

cerotic,  120 

citric,  92 

dihydroxystearic,  123 

elaidic,  121 

erucic,  121 

formic,  68,  114,  115 

gluconic,  67 

glycuronic,  77 

hypogaeic,  1 21 

isolinolenic,   122 

lactic,  68 

lanoceric,  123 

lauric,  119 

levulinic,  78 

lignoceric,  120 

linoleic,  122 

linolenic,  122 

linusic,   123 

mannosaccharic,  79 

melissic,  120 

myristic,  119 

mucic,  79 

oleic,  121 

palmitic,  120 

propionic,  114,  115 

ricinoleic,  123 


Acid,  saccharic,  79 

salicylic,  231 

sativic,  123 

stearic,  120 

tartaric,  67 

tartronic,  67,  68 

trioxyglutaric,  79 

valerianic,  114 
Acid  albumin,  200 
Acid  equivalent,  44 

Acidity,  112,  129,  149,  174,  194,  212,  233 
Acid  number,  129,  149 
Acids,   112,   114,  116,  119,  123,  163,  171 
Acme  sugar,  97 
Acrolein,  36 

Adamkiewicz  reaction,  204 
Addition  reactions,  47,  55 
Albuminates,  200 
Albuminoids,  202 
Albumins,  199 
Albumoids,  202 
Alcohol,  24,  29,  33,  35,  36,  52,  54,  55, 

57,  237 

Alcohols,  23,  33,  139,  144,  237 
Aldehydes,  46,  48,  52 
Allihn  method,  100 
Almond  oil,  147 
Alpha-naphthol  reaction,  76 
Alumina  cream,  87 
Ammonia  in  milk,  234 
Ammoniacal  silver  nitrate,  46 
Amyl  alcohols,  35 
Angular  rotation,  82,  84 
Anilin  acetate,  77 
Antifluorescents,   197 
Antiseptics,  2,  228 

Arabinose,  58,  61,  65,  71,  79,  83,  107 
Arachis  oil,  120,  121,  147,  152,  164 
Ash,  i,  4,  44,  93,  97,  112,  116,  211,  221 
Atmospheric  oxidation,  161 

Barfoed  method,  75 
Basic  lead  acetate,  87 
Beef  fat,  146 
Beeswax,  144,  146 

239 


240 


SUBJECT    INDEX. 


Beets,  94,  95,  96 

Benedikt  and  Cantor  method,  39 

Benzaldehyde,  47,  55 

Berthelot  method,  16,  19 

Beta-naphthol  test,  51 

Bioses,  see  disaccharides. 

Biot's  formula,  81 

Bisulphite  reaction,  47 

Birotation,  83 

Biuret  reaction,  204 

Blasdale  test,  155 

"Blown"  oils,  161,  190 

Boiling  point  method,  32 

Borax,  232 

Boric  acid,  232 

Boudouin's  test,  153 

Bran,  61,  105 

Breadstuff's,  208 

Brewer's  sugar,  96 

Bromine  substitution  number,  158 

Briigelmann  method,  15 

Burning  point,  196 

Butter,  1 66 

Butter  fat,  119,  120,  130,  146,  168 

Butter  oil,  176 

Butyro  refractometer,  142 

Cacao  butter,  146 
Cane  sugar,  see  sucrose. 
Carbohydrates,  58,  61,  62,  76,  8l,  85, 

99,  IGO 

Carbonaceous  residue,  36,  44 
Carbonates,  6,  112,  233 
Carbonyl,  47,  58,  64 
Carboxyl,  1 12 
Carius  method,  3,  14 
Carnaiiba  wax,  120,  144,  146 
Casein,  202,  207,  225 
Castor  oil,  123,  126,  139,  147 
Cellulose,  58,  61,  62,  63,  85,  107 
Cereals,  60,  61,  107,  108,  208 
Chilling  point,  195,  196 
Chlorides,  5,  44,  97 
Cholesterol,  145,  158 
Chromo-proteids,  201 
Claesson's  method,  15 
Clarification,  83,  88,  90 
Clerget  method,  80,  91 
Climax  sugar,  97 
Cocoanut  oil,  119,  120,  146,  176 


86, 


Codliver  oil,  147 

Cold  test,  195 

Collagen,  202 

Colophony,  146 

Color  reactions,  34,  46,  47,   50,   51,   148, 

158,  203,  228,  230,  232,  233 
Colza,  see  rape. 
Commercial  values  of  oils,  163 
Compensation  apparatus,  84 
Concrete,  94 
Condensation  methods,  46,  54,  63,  66,  76, 

77 

Confectionery,  1 10 
"Constants,"    127,   136,    146,    147,    I49? 

161,  163,  175 

Copper  reduction,  67,  68,  69,  74,  75 
Corn  oil,  147,  155,  159,  164 
Corn  sirup,  97 
Cossettes,  95 
Cottonseed  oil,  147,   150,    159,   161,  164, 

237 

Cottonseed  stearin,  146 
Crismer's  test,  173 
Critical  temperature  of  solution,  173 
Cyanide  method,  55 
Cylinder  oils,  198 

Defren's  method,  72 

Dextrin,  58,  60,  62,  81,  83,  98,  103,  107 

Dextrose,  58,  62,  66,  67,  68,  71,  72,  75, 

79,  80,  82,  83,  96,  98,  100,  109, 

in,  237 

Diabetic  sugar,  see  dextrose. 
Diastase,  59,  60,  61,  103,  104 
Dichromate,  see  oxidation  methods. 
Dihydrazone,  see  osazone. 
Dinitrobenzoate  test,  25 
Disaccharides,  58,  59,  62,  80 
Distilled  liquors,  29,  48 
Distillation  methods,  26,  37 
Double  dilution,  88,  225 
Drying  oils,  122,  157 
Dumas  method,  3 
Dyer  method,  9,  10 
Dynamite  glycerin,  44 

Ebullioscope,  32 

Elaidin,  121 

Enzymes,  59,  60,  61,  85,  103,  108 

Ester  number,  129 


SUBJECT   INDEX. 


241 


Ethyl  alcohol,  see  alcohol. 

iodide,  34 
Exposed  oils,  161 
Extract  (in  vinegar),  116 
Extractives  (nitrogenous),  202,  203 

Fat,  125,  182,  208,  209,  219,  222 
Fatty  acids,  see  acids. 
Fatty  oils,  125,  147,  148 
Fehling  method,  69 

solution,  67,  68,  69 
Fermented  liquors,  26,  40,  41 
Fermentation   products    (in    milk),    228, 

233,  234 
Fiber,  211 
Fire  test,  196 
Fish  oils,  147,  160 
Fixed  oils,  see  fatty  oils. 
Flame  reaction  (vinegar),   117 
Flash  test,  196 

Flour,  61,  105,  208,  212,  213,   214 
Fluorides,  233 
Food  preservatives,  48,  228 
Formal,  see  formaldehyde. 
Formaldehyde,  2,  47,  48,  228 
Formalin,  see  formaldehyde. 
Formol,  see  formaldehyde. 
Friction  tests,  197 
Fructose,  58,  59,  62,  68,  71,  75,  80,  82, 

109 

Fuchsin  test,  47 
Furfurol,  76,  77,  237 
Fusel  oil,  35,  237 

Galactan,  58,  59,  6l,  62,  79,  8l 
Galactosazone,  65 

Galactose,  58,   59,  60,  65,  71,  75,  79,  82 
Gallic  acid  test,  50 
Gallisin,  80 
Gelatin,  186,  202 
Gelatinoids,  202 
Gelatin  oils,  190 
Gerrard  method,  72 
Globulins,  200 
Glucosazone,  65 

Glucose,  58,  62,  66,  67,  68,  71,  72,  75, 
79,  82,  96,  97,  100,  109,  in,  237 
Glucoses,  58,  61,  64,  80 
Gluten  flour,  212,  214 
Glycerides,  125,  126 


Glycerin,  42,  44,  184,  237 
Glycerol,  36,  42,  44,  184,  237 
Glycogen,  58,  60,  62,  81,  83,  ill 
Glycol  (trimethylene),  43,  44 
Glyco-proteids,  201 
Gossypose,  see  raffinose. 
Graham  flour,  61,  105 
Grain,  60,  61,  208,  212,  213 
Grape  sugar,  see  glucose. 
Greases,  189 

Gums,  see  dextrin  and  pentosans. 
Gunning  method,  9,  10 

Halphen  test,  150 
Hanus  method,  131,  135 
Heat  of  combustion,  143,  147 
Hehner  method  (glycerol),  37 
(vinegar),  117 

number,  127,  130,  171,  175 
Hemicellulose,  81 
Hempseed  oil,  147,  157 
Henriques  method  of  saponification,  128 
Herzfeld  method,  92,  93 
Hexabromide  test,  160 
Hexoses,  58 
Honey,  no 

Hubl  method,  127,  130,  131 
Hydrazone,  63 
Hydrogen  peroxide  method,  53 

in  milk,  230 

Hydrolysis,  80,  91,  99,  loo,  105,  107 
Hydroxy-acids,  122,  123,  138,  140 
Hydroxyl,  23,  39 
Hydroxylamine,  46 

Immiscible  solvents,  191 

Index  of  refraction,    127,    141,   146,  147, 

149,  161,  175 
Infusion  of  malt,  104 
International  methods  of  sugar  analysis, 

88,92 

Inversion,  see  hydrolysis. 
Invert   sugar,  59,  66,  68,  71,  75,  83,  91, 

92,  109 

lodimetric  method,  52 
Iodine  absorption,  130,  146,  147,  161,  172 
Iodine  number,   130,  146,   147,  149,  161, 

172 

lodoform  test,  25 
Isomaltose,  80 


242 


SUBJECT   INDEX. 


Japan  wax,  146 
Jodlbauer  method,  13 

Ketones,  47,  58,  67 

Kjeldahl  method  (nitrogen),  8,  10,  237 

(sugar),  74,  75 
Koettstorfer  number,  127,  146,  147 

Lactic  fermentation,  104 

Lactometer,  218 

Lactosazone,  65 

Lactose,  58,  59,  61,  62,  64,  66,    71,  72, 

75,  79,  81,  83,  no,  215,  225 
Lam's  method,  34 
Lard,  120,  146,  176 
Lard  oil,  147,  155,  161 
Lead  acetate  (basic),  87 
Legler  method,  54 
Levulose,  58,  59,  62,  68,  71,  75,  80,  82, 

109 

Lieben's  test,  25 

Liebermann-Storch  reaction,  158 
Liebig's  fusion  method,  15,  1 6,  20 
Linseed  oil,  122,  147,  157,  161,  165 
Liquors,  26,  34,  40,  41,  237 
Lubricants,  189,  198 

Maize  oil,  147,  155,  159,  164 

Malt  infusion,  104 

Maltosazone,  65 

Maltose,  58,  59,   64,66,   71,  72,   75,  80, 

83,  98,  103,  108,  in 
Malt  sugar,  see  maltose. 
Mannose,  58,  59,  64,  79 
Maple  sirup,  94,  no 

sugar,  94,  no 
Margarine,  176 
Marquardt's  method,  35 
Massecuite,  94 
Maumene  test,  127,  136 

number,   127,  136,  147 
Mayrhofer  method,  106 
Meal,  212 
Meat,  207 
Melada,  94 

Melitriose,  see  raffinose. 
Melibiose,  60 

Melting  point,  127,  142,  146,  172 
Menhaden  oil,  147,  160 
Metals,  7 


Methyl  alcohol,  33,  34,  49,  54,  55,  57 

iodide,  34 

violet,  117 

Methylated  spirit,  33,  36 
Methylene-dibetanaphthol  test,  51 
Millon's  reaction,  204 
Milk,  215 
Milk  sugar,  58,  59,  61,  62,  64,  66,  71,  72, 

75,  79,  81,  83,  no,  215,  225 
Mineral  oil,  147,  157 
Moisture,  93,  no,  208 
Molasses,  93,  94,  no 
Molisch  reaction,  76 
Monosaccharides,  58,  61,  64,  80 
Monoses,  see  monosaccharides. 
Mucins,  201 
Mucoids,  20 1 
Multirotation,  83 
Mush  sugar,  94 
Mustard  oil,  121,  147 
Muter' s  method,  121,  123 

Neatsfoot  oil,  147 

Neumann  method,  21 

Nitrates,  13 

Nitric  acid  reaction  of  oils,  151,  155 

Nitro-compounds,  13,  42 

Nitrogen,  8,   10,  199,  202,  203,  206,  210, 

212,  224,  237 
Nucleo-albumins,  201 
Nucleo-proteids,  201 

Oatmeal,  105,  212 

Occurrence  of  carbohydrates,  58 
of  fatty  acids,  119 
of  fats  and  waxes,  146,  147 

Official  standards,  93,  94,  96,    118,   212, 
215,  227 

Oil,  almond,  147,  163 

arachis,  120,  1 21,  147,  152,  163,  164 
castor,  123,  126,  139,  147,  163 
cocoanut,  119,  120,  146,  176 
codliver,  147,  163 
colza,  see  rape, 
copac,  151 

corn,  147,  155,  159,  163,  164 
cottonseed,  147,   150,  159,   161,  163, 

164,  237 

hempseed,  147,  157 
lard,  147,  155,  161,  163 


SUBJECT   INDEX. 


243 


Oil,  linseed,  122,  147,  157,   161,  163,  165 

maize,  147,  155,  159,  163,  164 

menhaden,  147,  1 60,  163 

mineral,  147,  157,  163 

mustard,  121,  147 

neatsfoot,   147,  163 

oleo,  146,  176 

olive,  147,  148,  161,  163 

palm,  119,  146,  176,  178 

peanut,  120,  121,   147,  152,  163,  164 

poppy,  147,  155,  163,  165 

rape,  1 21,  147,  154,  163 

rosin,  147,  158,  163 

seal,  121,  147,  163 

sesame,  147,  153,  163,  164,  176 

sperm,   121,  147,  163 

sunflower,  147 

tung,  147,  157 

whale,  147,  163 

wood,  see  tung. 
Oils,  125,  126,  147,  148 
Oleomargarine,  176 
Oleo  oil,  146,  176 
Oleo-refractometer,  141 
Olive  oil,  147,  148,  161,  163 
Optical  rotation,  81,  82,  85,  1 1 8,  201 
Osazones,  64,  65,  237 
Osborne  method,  15,  18 
Ost  method,  72 

Oxidation  methods,  19,  33,  34,  35,  37,  46, 
52,  S3,  67,  69,  72,  74,  75,  79,  114 
Oxidized  oils,  161,  165 
Oxyacetonitril,  56 
Oxymethylene,  49 
Palm  oil,  119,  146,  176,  178 
Paraffin,  146 
Parafonnaldehyde,  49 
Paraldehyde,  54,  55,  57 
Paranucleo-proteids,  201 
Pavy  method,  72 

Peanut  oil,  120,  121,  147,  152,  163,  164 
Pentosans,   58,  61,  62,  63,  77,  81,    104, 

107,  237 

Pentoses,  58,  61,  77 
Peptones,  200,  204 
Phenols,  47,  182 
Phenylhydrazine,  46,  63,  77 
Phloroglucin,  77,  78 
Phosphates  in  ash,  6 
Phosphorus,  6,  20,  22 


Phytosterol,  145 

Phytosteryl  acetate  test,  155,  173 

Piotrowski  reaction,  204 

Polariscope,  84,  86 

Polarization,  81,  88,  89,  90 

Polarized  light,  81,  85 

Polysaccharides,  58,  61,  62,  63,  80 

Poppy  oil,  147,  155,   165 

Potassium  cyanide  method,  55 

Preparation  of  samples,  2,  180,  218 

Preservation  of  samples,  2,  218 

Proof  spirit,  36 

Proteids,  199,  203,  205,  206,  210,  224 

Protein,  203 

Proteoids,  202,  203 

Proteoses,  200,  204 

Proximate  analysis,  I 

Pyknometer,  30,  31 

Quartz  compensation,  84 

Raffinose,  58,  59,  79,  81,  83,  no 

Rancidity,  174,  178 

Rape  oil,  121,  147,  154 

Raw  sugar,  86 

Rectified  spirit,  36 

Reducing  sugars,  ,67,  92,  107,  237 

Refraction  (index  of),  127,  141,  146,  147 

149,  161,  175 
Refractometers,  141 
Reichert-Meissl   number,   127,    130,    166, 

175 

Reichert-Wollny  number,  168,  175 
Reimann  sinker,  42 
Relations  between  carbohydrates,  58 

"constants"    of  fats,   127,  129, 
135,   143,    146,  147,  150,  156, 

161,  175 

constituents  of  milk,  215,  227 
Renard-Tolman  test,  152 
Resorcin  test,  50 
"  Reversion,"  80 
Rice,  212 
Roese  method,  35 
Romijn  method,  56 
Rosin,  146,  158 
Rosin  oil,  147,  158 
Rotation-dispersion,  82 
Rotation  (optical),  8l,  85,  118,  201 
Rotating  power,  81,  82,  85,  118,  201 


244 


SUBJECT    INDEX. 


Saccharimeter,  84,  86,  89 
Saccharose,  see  sucrose. 
Sachsse  method,  loo 
Salad  oils,  148 
Salicylates,  231 
Salicylic  acid,  231 
Saliva,  103 

Sampling,  I,  180,  217 
Saponification  equivalent,  129 

number,    127,    129,     146,    147,    171, 

175 

Sauer's  method,  15 
Sausages,  105 
Schweitzer's  reagent,  63 
Scovell  tube,  217 
Seal  oil,  121,  147 
Separation  of  acids,  114,  116,  123 
Sesame  oil,  147,  153,  164,  176 
Sidersky  method,  72 
Sitosterol,  145 
Sirup,  94,  no 
Soap,  1 80 

Soap  solutions  of  oils,  155 
Soda-lime  method,  8 
Sodium  peroxide  method,  1 8,  237    . 
Soldani  method,  72 
Solids  in  milk,  215,  220,  227 

in  vinegar,  116,  118 
Solubilities  of  carbohydrates,  61,  62 

of  fats,  126,  127 
Sources  of  carbohydrates,  58 

of  oils,  fats  and  waxes,  146,  147 
Soxhlet  apparatus,  181,  182,  222 
Specifications,  36,  44,  189,  195,  196  (see 

also  standards). 

Specific  gravity,    27,  29,  34,  36,  42,43, 
49,   127,   140,   146,  147,  149,  i6i» 
171,  175,  218 
Specific  refractive  power,  161 

rotatory  power,  81 
Spermaceti,  120,  144,  146 
Sperm  oil,  121,  147 
Sprengel  tube,  32,  42 
Standards,  36,  93,  94,  96,  118,  212,  215, 

227 

Starch,  58,  60,   62,  63,  Si,  83,  100,  105, 
106,  107 

sugar,  see  glucose. 
Storch  reaction,  158 
Substitution  number,  158 


Sucrose,  58,  62,  63,  64,  68,  80,  83,  86, 

89,  90,  91,  93.  94,  107,  109 
Sugar,  see  sucrose. 
Sugar  beets,  94,  95 

cane,  94,  95 
Sugar,  malt,  see  maltose. 

milk,  see  lactose. 
Sulphur,  6,  14,  237 
Sunflower  oil,  147 

Tallow,  146 
Tannin,  83 
Temperature  corrections,  30,  43,  141,  142 

reaction,  127,  136,  147,  149,  161 
Tetroses,  58 

Thorpe  and  Holmes  method,  34 
Thymol,  76 
Tinctures,  29 
Titer  test,  127,  142 
Tocher's  test,  154 
Tollen's  aldehyde  reagent,  46 
Treacle,   97 

Trirnethylene  glycol,  43 
Trioxymethylene,  49 


Tryptophan  reaction,  204 
Tung  oil,  147,  157 
TwitchelPs  method,  158 

Ultimate  analysis,  I,  2,  237 
Unification  of  methods,  88 
Unknown  oils,  162 
Unsaponifiable*matter,  144,  I  Si,  182 

oils,  190 

Unsaturated  acids,*i2i,  127 
Urine,  67,  in 

Values  of  oils,  163,  see  also  "  constants. 

Vanillin,  47 

Ventzke  scale,  84,  85,  86,  87 

yillavecchia's  test,  154 

Vinegar,  115,  117 

Viscosimeters,  156,  192 

Viscosity,  127,  143,  155,  192,  198 

number,  156,  161 
Vitellin,  202 

Water  determination,  93,  1  10,  208 
Waxes,  125,  139,  144 
Whale  oil,  147 


SUBJECT   INDEX. 


245 


Wheat,  6l,  105 
Wijs  method.  131,  134,  135 
Wiley  ebullioscope,  32 
method  (alcohol),  32 

(glucose),  97 
Wilfarth  method,  9 
Wines.  26,  40,  41 
Westphal  balance,  140 
Woodgum,  see  pentosans. 


Wood  naphtha,  36 
spirit,  36,  49 
vinegar,  115 

Xanthoproteic  reaction,  203 

Xylan,  6 1 

Xylosazone,  65 

Xylose,  58,  61,  71,  79,  83,  107 

Zero  point  of  polariscope,  86 


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NOV  29   1944 

.  MAY   S  1947 

-      '^1 

; 

5                               ,     : 

•2N«g'6ABS 

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